Nucleic acids encoding aggrecan degrading metallo proteases

ABSTRACT

The invention is directed to the family of aggrecan degrading metallo proteases (ADMPs) that exhibit the ability to cleave the aggrecan core protein between amino acid residues Glu 373 -Ala 374 . The invention encompasses the nucleic acids encoding such enzymes, processes for production of recombinant ADMPs, compositions containing such enzymes, and the use of these enzymes in various assays and for the development of novel inhibitors for use as therapies for diseases involving aggrecanase-mediated degradation of cartilage or other aggrecanase-associated diseases.

CROSS REFERENCE TO EARLIER FILED APPLICATION

This application is a division of U.S. Ser. No. 09/122,126, filed Jul.24, 1998 which now is a U.S. Pat. No. 6,451,575 and claims the benefitof U.S. Provisional Application No. 60/053,850 filed on Jul. 25, 1997and U.S. Provisional Application No. 60/055,836 filed on Aug. 15, 1997and U.S. Provisional Application No. 60/062,169 (unknown at filing),filed on Oct. 16, 1997.

FIELD OF THE INVENTION

The invention is directed to the family of proteins that exhibitaggrecanase activity, the nucleic acids encoding such enzymes, processesfor production of recombinant aggrecanases, compositions containing suchenzymes, antibodies raised against these enzymes, and the use of theseenzymes or antibodies in various assays and therapies.

BACKGROUND OF THE INVENTION

Aggrecan is the major proteoglycan of cartilage and provides this tissuewith its mechanical properties of compressibility and elasticity. Inarthritic conditions one of the earliest changes observed in cartilagemorphology is the depletion of aggrecan [Mankin et al. (1970) J. BoneJoint Surg. 52A, 424-434], which appears to be due to an increased rateof degradation.

The aggrecan molecule is composed of two N-terminal globular domains, G1and G2, which are separated by an approximately 150 residueinterglobular domain (IGD), followed by a long central glycosaminoglycan(GAG) attachment region and a C-terminal globular domain, G3 [Hardinghamet al. (1992) in Articular Cartilage and Osteoarthritis: Aggrecan, TheChondroitin Sulfate/Keratin Sulfate Proteoglycan from Cartilage(Kuettner et al.) pp. 5-20, Raven Press, New York and Paulson et al.(1987) Biochem. J. 245, 763-772]. These aggrecan molecules interactthrough the G1 domain with hyaluronic acid and a link protein to formlarge molecular weight aggregates which are trapped within the cartilagematrix [Hardingham et al. (1972) Biochim. Biophys. Acta 279, 401-405,Heinegard et al. (1974) J. Biol. Chem. 249, 4250-4256, and Hardingham,T. E. (1979) Biochem. J. 177, 237-247]. Loss of aggrecan from cartilagein arthritic conditions involves proteolytic cleavage of the aggrecancore protein within the IGD, producing a N-terminal G-1 fragment thatremains bound to hyaluronic acid and the link protein within the matrix,releasing a large C-terminal GAG-containing aggrecan fragment thatdiffuses out of the cartilage matrix. Loss of the C-terminal fragmentresults in cartilage deficient in its mechanical properties. Thisdeficiency arises because the GAGs are the components of aggrecan thatimpart the mechanical properties to the molecule through their highnegative charge and water binding capacity.

Two major sites of proteolytic cleavage have been identified within theIGD, one between amino acid residues Asn³⁴¹-Phe³⁴² and the other betweenamino acid residues Glu³⁷³-Ala³⁷⁴. Although G1 fragments formed bycleavage at the Asn³⁴¹-Phe³⁴² site and at the Glu³⁷³-Ala³⁷⁴ site havebeen identified within articular cartilage [Flannery et al. (1992) J.Biol. Chem. 267, 1008-1014], the N-terminus identified on the largeGAG-containing aggrecan C-terminal fragments in synovial fluids ofpatients with osteoarthritis [Sandy et al. (1992) J. Clin. Invest. 69,1512-1516], inflammatory joint disease [Lohmander et al. (1993)Arthritis Rheum. 36, 1214-1222] and in the media from cartilage explantand chondrocyte cultures stimulated with interleukin-1 or retinoic acid[Sandy et al. (1991) J. Biol. Chem. 266, 8198., Sandy et al. (1991) J.Biol. Chem. 266, 8683-8685., Loulakis et al. (1992) Biochem. J. 264,589-593., Ilic et al. (1992) Arch. Biochem. Biophys. 294, 115-122., Larket al. (1995) J. Biol. Chem. 270, 2550-2556.] was ARGSVIL, indicatingthat they were formed by cleavage between amino acid residuesGlu³⁷³-Ala³⁷⁴. These observations suggest that cleavage at this site maybe responsible for cartilage degradation.

Although many matrix metalloproteases (MMP-1, -2, -3, -7, -8,-9 and 13)have been shown to cleave in vitro at the Asn³⁴¹-Phe³⁴² site, digestionof aggrecan with a number of these purified proteases has not resultedin cleavage at the Glu³⁷³-Ala³⁷⁴ site [Fosang et al. (1992) J. Biol.Chem. 267, 19470-19474., Flannery et al. (1992) J. Biol. Chem. 267,1008-1014., Fosang et al. (1993) Biochem. J. 295, 273-276., Fosang etal. (1996) FEBS Lett. 380, 17-20., Flannery et al. (1993) Orthop. Trans.17, 677., and Fosang et al. (1994) Biochem. J. 305, 347-351]. Therefore,cleavage at this site has been attributed to a novel, proteolyticactivity, “aggrecanase”.

In addition to the Glu³⁷³-Ala³⁷⁴ bond within the interglobular domain ofaggrecan, four potential aggrecanase-sensitive sites have beenidentified within the C-terminus of the aggrecan core protein [Loulakiset al. (1992) Biochem. J. 264, 589-593. and Sandy et al. (1995) ActaOrhtop Scand (Suppl 266) 66, 26-32]. Although cleavage at these siteswhich are not within the interglobular domain would not be expected torelease the major portion of the aggrecan molecule from the matrix, theymay be involved in earlier processing of aggrecan within the matrix.

SUMMARY OF THE INVENTION

The invention encompasses a novel family of biologically active aggrecandegrading metallo proteases (“ADMP”) capable of cleaving the aggrecanmonomer core protein at the Glu³⁷³-Ala³⁷⁴ aggrecanase site, as isolatedand purified polypeptides. An object of the invention covers novelsequences of nucleic acids which encode for members of the ADMP family,and to expression vectors containing cDNA which encodes for novelmembers of the ADMP family. Another object of the invention is hostcells that have been transfected or transformed with expression vectorswhich contain cDNA that encodes for the ADMP family of polypeptides, andprocesses for producing members of the ADMP family by culturing suchhost cells under conditions conducive to expression of an ADMP. Anotherobject of the invention is probes containing nucleic acid sequences thathybridize to a native ADMP nucleotide sequence and the use of theseprobes for detection of message for an ADMP in biological samples. Afurther object of the invention is antibodies raised against an ADMP,which may be created as a result of the purification and isolation ofmembers of the ADMP family and the use of such antibodies for thedetection of ADMPs in biological samples. Assays utilizing an ADMP toscreen for its potential inhibitors are another object of thisinvention. Members of the ADMP family used to design novel inhibitors ofproteases exhibiting aggrecanase activity are also part of the instantinvention.

Members of the ADMP family are capable of cleaving the aggrecan monomercore protein at the Glu³⁷³-Ala³⁷⁴ site, but do not readily cleaveaggrecan at the Asn³⁴¹-Phe³⁴², MMP-sensitive cleavage site, and thezymogen form of the protein consists of the following domains: apropeptide domain containing a furin site, a metalloprotease domain, adisintegrin-like domain and a thrombospondin homologous domain.

As used herein, the term “zymogen” refers to the latent, full-lengthprotein synthesized by the cells and further processed to acatalytically active form, the term “propeptide domain” refers to theN-terminal region of the molecule which contains a cysteine residueinvolved in latency of the protein, the term “furin cleavage site”refers to a region of the molecule containing a tetra basic sequence ofamino acids susceptible to cleavage by furin or furin-like proteases,the term “metalloprotease domain” refers to a region of the moleculewhich contains a zinc-binding motif with the consensus sequence,HExxHxxGxxH, responsible for the catalytic activity of the protein, theterm “disintegrin-like domain” refers to a region of the molecule whichexhibits sequence similarity to the disintegrin family of anti-coagulantpeptides found in snake venoms, which are characterized by a highcysteine content and have the ability to disrupt cell-matrixinteraction, and the term “thrombospondin homologous domain” refers to aregion of the molecule containing one or more thrombospondin type 1(TSP1) motifs with sequence homologous to the amino acid sequence ofTSP1 repeats which are conserved in thrombospondin 1 and 2 and have beenimplicated in the interaction of thrombospondin with sulfatedglycoconjugates such as heparin and heparan sulfate.

The first isolated and purified ADMP family member according to theinvention, referred to as “ADMP-1”, has a molecular weight between about50 kD and about 98 kD as determined by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). More specifically, the isolated activeADMP-1 was found to have a molecular weight of approximately 67 kD asdetermined by SDS-PAGE. The isolated and purified metalloprotease of theinvention is capable of cleaving the aggrecan monomer core protein atthe Glu³⁷³-Ala³⁷⁴ site, but does not readily cleave aggrecan at theAsn³⁴¹-Phe³⁴², MMP cleavage site and consists of the following domains:a propeptide domain containing a furin site, a metalloprotease domain, adisintegrin-like domain and a thrombospondin homologous domain. The cDNAsequence of ADMP-1 is shown in SEQ ID NO:1. The isolated and purifiedADMP-1 zymogen constitutes amino acids 1-837 of SEQ ID NO:2 and has amolecular weight of about 98 kDa as determined by SDS-PAGE.

The second isolated and purified aggrecanase according to the invention,referred to as “ADMP-2”, has a molecular weight between about 45 kD andabout 93 kD as determined by SDS-PAGE. More specifically the isolatedactive ADMP-2 was found as four forms of the same gene product havingmolecular weights of approximately 50 kD, 54 kD, 62 kD and 64 kD asdetermined by SDS-PAGE. This second isolated and purifiedmetalloprotease of the invention is capable of cleaving the aggrecanmonomer core protein at the Glu³⁷³-Ala³⁷⁴ site, but does not readilycleave aggrecan at the Asn³⁴¹-Phe³⁴², MMP cleavage site. The cDNAsequence of ADMP-2 is shown in SEQ ID NO:14. The isolated and purifiedADMP-2 zymogen constitutes amino acids 1-930 of SEQ ID NO:15 and has amolecular weight of about 93 kDa as determined by SDS-PAGE.

The instant invention describes a method for treating a mammal having adisease characterized by an overproduction or an upregulated productionof an ADMP. This treatment involves administration of a compositioncontaining an efficacious amount of a compound that inhibits theproteolytic activity of members of the ADMP family. These enzymesinclude, but are not limited to, those containing the sequence of aminoacids 1-837 of SEQ ID NO:2 or the sequence of amino acids from 1-930 ofSEQ ID NO:15.

The potency of compounds in inhibiting soluble, active ADMP activity inconditioned media from interleukin-1-stimulated bovine nasal cartilage,correlates with their potency in inhibiting cartilage aggrecan cleavageand release from cartilage. This ADMP activity is monitored using anenzymatic assay employing purified aggrecan substrate. Specific productsof aggrecanase-mediated cleavage are detected by Western analysis usingthe monoclonal neoepitope antibody, BC-3 [Hughes et al., Biochem. J.306:799-804 (1995)]. This antibody recognizes the newly-formedamino-terminal sequence NH₂-ARGSVIL on fragments produced by cleavage atthe Glu³⁷³-Ala³⁷⁴ aggrecanase site. The term “neoepitope antibody”refers to an antibody which specifically recognizes a new N-terminalamino acid sequence or new C-terminal amino acid sequence generated byproteolytic cleavage but does not recognize these same sequences ofamino acids when they are present within the intact protein.

Aggrecanase-mediated degradation of cartilage aggrecan has beenimplicated in osteoarthritis, joint injury, reactive arthritis, acutepyrophosphate arthritis (pseudogout), psoriatic arthritis and juvenilerheumatoid arthritis. Inhibitors of ADMPs would prevent cleavage of theaggrecan core protein, thereby decreasing the loss of aggrecan from thecartilage. The instant invention contains such an embodiment and alsodescribes a method of inhibiting the cleavage of aggrecan in cartilageof a mammal by administering an efficacious amount of a compound thatinhibits the aggrecanase proteolytic activity of an enzyme of the ADMPfamily. These enzymes include, but are not limited to, those containingthe sequence of amino acids from 1-837 of SEQ ID NO:2 or the sequence ofamino acids from 1-930 of SEQ ID NO:15.

Inhibitors of members of the ADMP family would be of significantclinical utility and could be potential therapeutic agents for treatingthe aggrecanase-related disorders cited above. ADMP inhibitors also haveclinical utility for the treatment of other conditions characterized byover-production or up-regulated production of an ADMP. Isolation andpurification of ADMPs would provide a significant advancement in thetreatment of aggrecanase-associated diseases and in the effort todevelop inhibitors of these enzymes.

DETAILED DESCRIPTION OF THE INVENTION

Isolated cDNAs encoding human ADMPs are disclosed in SEQ ID NO:1 and SEQID NO:14. This discovery of cDNAs encoding human ADMPs enablesconstruction of expression vectors comprising nucleic acid sequencesencoding ADMPs, host cells transfected or transformed with theexpression vectors, biologically active human ADMPs as isolated andpurified proteins, and antibodies immunoreactive with ADMPs.

Isolated and purified ADMP polypeptides according to the invention areuseful for detecting the aggrecanase-inhibiting activity of a molecule.In such a method involving routine and conventional techniques, amolecule of unknown aggrecanase-inhibiting activity is mixed with asubstrate and incubated with an ADMP polypeptide. The extent ofsubstrate cleavage then can be determined using a neoepitope antibody todetect cleavage fragments generated by specific cleavage at theGlu³⁷³-Ala³⁷⁴ bond.

In addition, ADMP polypeptides according to the invention are useful forthe structure-based design of an aggrecanase inhibitor. Such a designwould comprise the steps of determining the three-dimensional structureof such ADMP polypeptide, analyzing the three-dimensional structure forthe likely binding sites of substrates, synthesizing a molecule thatincorporates a predictive reactive site, and determining theaggrecanase-inhibiting activity of the molecule.

Antibodies immunoreactive with ADMPs are now made available through theinvention. Such antibodies may be useful for inhibiting aggrecanaseactivity in vivo and for detecting the presence of an ADMP in a sample.As used herein, the term “ADMP” refers to a family of polypeptides thatare capable of cleaving the aggrecan core protein at the Glu³⁷³-Ala³⁷⁴bond, but do not readily cleave at the Asn³⁴¹-Phe³⁴² bond and consist ofthe following domains: a propeptide domain containing a furin site,followed by a metalloprotease domain, followed by a disintegrin-likedomain, followed by a thrombospondin homologous domain, wherein thepolypeptide is either a native or recombinant polypeptide.

The ADMP family encompasses, but is not limited to, proteins having theamino acid sequence 1 to 837 of SEQ ID NO:2 or the sequence of aminoacids from 1-930 of SEQ ID NO:15, as well as those proteins having ahigh degree of similarity (at least 80% homology) with the amino acidsequence 1-837 of SEQ ID NO: 2 or the sequence of amino acids from 1-930of SEQ ID NO:15 and which proteins are biologically active. In addition,ADMP refers to the biologically active gene products of the nucleotides405-2919 of SEQ ID NO: 1 and to the biologically active gene products ofthe nucleotides 121-2910 of SEQ ID NO:14. Further encompassed by theterm “ADMP” are the truncated proteins that retain biological activity.Truncated versions are those having less of the C-terminal or N-terminalportion of the protein or that comprise substantially all of thecatalytic domain, i.e., amino acids 212-431 of SEQ NO:2 or amino acids262-479 of SEQ ID NO:15.

The first isolated and purified active aggrecanase according to theinvention, referred to as “ADMP-1”, has a molecular weight between about50 kD and about 98 kD as determined by sodium doecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Morespecifically, ADMP-1 was found to have a molecular weight ofapproximately 67 kD as determined by SDS-PAGE. The second isolated andpurified active aggrecanase according to the invention, referred to as“ADMP-2”, has a molecular weight between about 45 kD and about 93 kD asdetermined by SDS-PAGE. More specifically, active ADMP-2 was present asfour forms of the same gene product found to have molecular weights ofapproximately 50 kD, approximately 54 kD, approximately 62 kD andapproximately 64 kD as determined by SDS-PAGE.

The term “isolated” as used herein, means that an ADMP is essentiallyfree from association with other proteins or polypeptides, for exampleas a purification product of recombinant host cell culture or as apurified product from a non-recombinant source. The term “substantiallypurified” as used herein, refers to a mixture that contains an ADMP andis essentially free from association with other proteases, and whichsubstantially purified ADMP retains biological activity. The term“purified ADMP” means that the ADMP is present in a cell-free system.The term “biologically active” as it refers to an ADMP, means that theADMP is capable of cleaving the aggrecan core protein at theGlu³⁷³-Ala³⁷⁴ bond.

This invention provides a nucleic acid molecule encoding ADMP-1 (SEQ IDNO:1) and ADMP-2 (SEQ ID NO:14). Examples of nucleic acid molecules areRNA, cDNA or isolated genomic DNA molecules. One means of isolating anADMP is to probe a cDNA or genomic library with a natural or artificialDNA probe derived from the ADMP-1 or ADMP-2 cDNA. DNA probes derivedfrom the ADMP-1 or ADMP-2 cDNA can be used to obtain complementary cDNA,RNA or genomic clones from human, mammalian or other sources, usingmethods known in the art (such as those outlined in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989)).

A “nucleotide sequence” refers to a polynucleotide molecule in the formof a separate fragment or as a component of a larger nucleic acidconstruct, that has been derived from DNA or RNA isolated at least oncein substantially pure form (i.e., free of contaminating endogenousmaterials and in a quantity or concentration enabling identification,manipulation, and recovery of its component nucleotide sequences bybiochemical methods (such as those outlined in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989)). Such sequences arepreferably provided in the form of an open reading frame uninterruptedby internal non-translated sequences, or introns, that are typicallypresent in eukaryotic genes. Sequences of non-translated DNA may bepresent 5′ or 3′ from an open reading frame, where the same do notinterfere with manipulation or expression of the coding region.

The term “aggrecan degrading metallo protease” (“ADMP”) as referred toherein, means a polypeptide substantially homologous to a native ADMPwhich is biologically active, and an amino acid sequence different fromthat of the native ADMP (human, bovine, canine, murine or other species)because of one or more deletions, insertions or substitutions. The termincludes a variant sequence wherein the variant amino acid sequencepreferably is at least 80% identical to a native ADMP amino acidsequence. The percent identity may be determined, for example, bycomparing sequence information using the GAP computer program, version6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilizes the alignment method of Needleman andWunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman(Adv. Appl. Math 2:482, 1981). The preferred default parameters for theGAP program include: (1) a unary comparison matrix (containing a valueof 1 for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

“Variants” as referred to herein comprise conservatively substitutedsequences, meaning that a given amino acid residue is replaced by aresidue having similar physiochemical characteristics. Conservativesubstitutions are well known in the art and include substitution of onealiphatic residue, such as Ile, Val, Leu, or Ala for another, orsubstitutions of one polar residue for another, such as between Lys andArg, Glu and Asp, or Gln and Asn. Conventional procedures and methodscan be used for making and using such variants. Other such conservativesubstitutions, for example, substitutions of entire regions havingsimilar hydrophobicity characteristics, are well known and routinelyperformed. Naturally occurring ADMP variants are also encompassed by theinvention. Examples of such variants are proteins that result fromalternate mRNA splicing events or from proteolytic cleavage of an ADMP,wherein the aggrecanase proteolytic property is retained. Alternatesplicing of mRNA may yield a truncated but biologically active ADMPs.Variations attributable to proteolysis include, for example, differencesin the N- or C-termini upon expression in different types of host cells,due to proteolytic removal of one or more terminal amino acids from theADMP.

As stated above, the invention provides isolated and purified, orhomogeneous, ADMP polypeptides, both recombinant and non-recombinant.Variants and derivatives of native ADMPs that retain the desiredbiological activity may be obtained by mutations of nucleotide sequencescoding for native ADMP polypeptides. Alterations of the native aminoacid sequence may be. accomplished by any of a number of conventionalmethods. Mutations can be introduced at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed procedures can be employed toprovide an altered gene wherein predetermined codons can be altered bysubstitution, deletion or insertion. Exemplary methods of making thealterations set forth above are disclosed by Walder et al. (Gene 42:133,1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods,Plenum Press, 1981); Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985);Kunkel et al. (Methods in Enzymol. 154:367, 1987); and Mark et al.(Proc. Natl. Acad. Sci. USA 18:5662, 1984) all of which are incorporatedby reference.

An ADMP may be modified to create ADMP derivatives by forming covalentor aggregative conjugates with other chemical moieties, such as glycosylgroups, polyethylene glycol (PEG) groups, lipids, phosphate, acetylgroups and the like. Covalent derivatives of an ADMP may be prepared bylinking the chemical moieties to functional groups on the ADMP aminoacid side chains or at the N-terminus or C-terminus of an ADMPpolypeptide. Other derivatives of an ADMP within the scope of thisinvention include covalent or aggregative conjugates of an ADMP or itsfragments with other proteins or polypeptides, such as by synthesis inrecombinant culture as N-terminal or C-terminal fusions. For example,the conjugate may comprise a signal or leader polypeptide sequence (e.g.the a-factor leader of Saccharomyces) at the N-terminus of an ADMPpolypeptide. The signal or leader peptide co-translationally orpost-translationally directs transfer of the conjugate from its site ofsynthesis to a site inside or outside of the cell membrane or cell wall.

ADMP polypeptide conjugates can comprise peptides added to facilitatepurification and identification of the ADMP. Such peptides include, forexample poly-His or the antigenic identification peptides described inHopp et al., Bio/Technology. 6:1204, 1988. The term “ADMP derivative”refers to an ADMP polypeptide conjugated with a chemical moiety, otherproteins or polypeptides encompassing, but not limited to, glycosylgroups, polyethylene glycol (PEG) groups, lipids, phosphate, acetylgroups, poly-His peptides, antigenic-identification peptides, signalpeptides or leader peptides.

The invention further includes ADMP polypeptides with or withoutassociated native-pattern glycosylation. An ADMP expressed in yeast ormammalian expression systems (e.g., COS-7 cells) may be similar to orsignificantly different from the native ADMP polypeptide in molecularweight and glycosylation pattern, depending upon the choice ofexpression system. Expression of ADMP polypeptides in expressionsystems, such as E. coli, provides non-glycosylated molecules. Glycosylgroups may be removed through conventional methods, in particular thoseutilizing glycopeptidase. In general, a glycosylated ADMP may beincubated with a molar excess of glycopeptidase.

Equivalent DNA constructs that encode various additions or substitutionsof amino acid residues or sequences, or deletions of terminal orinternal residues or sequences not needed for biological activity, areencompassed by the invention. For example, N-glycosylation sites in theADMP extracellular domain can be modified to preclude glycosylation,allowing expression of a reduced carbohydrate analog in mammalian andyeast expression systems. N-glycosylation sites in eukaryoticpolypeptides are characterized by an amino acid triplet Asn-X-Y, whereinX is any amino acid except Pro and Y is Ser or Thr. Appropriatesubstitutions, additions or deletions to the nucleotide sequenceencoding these triplets will result in prevention of attachment ofcarbohydrate residues at the Asn side chain. Alteration of a singlenucleotide, chosen so that Asn is replaced by a different amino acid forexample, is sufficient to inactivate the N-glycosylation site. Knownprocedures for inactivating N-glycosylation sites in proteins includethose described in Larsen et al. (J. Biol. Chem. 263:1023, 1988), Hansenet al. (J. Biol. Chem. 263:15713, 1988) and Larsen et al. (Blood73:1842, 1989), hereby incorporated by reference.

In another example, sequences encoding Cys residues that are notessential for biological activity can be altered to cause the Cysresidues to be deleted or replaced with other amino acids, preventingformation of incorrect intramolecular disulfide bridges uponrenaturation. Other equivalents may be prepared by modification ofadjacent dibasic amino acid residues to enhance expression in yeastsystems in which protease activity is present.

Nucleic acid sequences within the scope of the invention includeisolated DNA and RNA sequences that hybridize to the native ADMPnucleotide sequences disclosed herein or those of other members of theADMP family under conditions of moderate or high stringency, and whichencode a biologically active ADMP. Conditions of moderate stringency, asknown to those having ordinary skill in the art, and as defined bySambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1,pp. 101-104, Cold Spring Harbor Laboratory Press, (1989), include use ofa prewashing solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) andhybridization conditions of about 50° C.-60° C., 5×SSC, overnight,preferably 55° C. Conditions of high stringency include highertemperatures of hybridization and washing. The skilled artisan willrecognize that the temperature and wash solution salt concentration maybe adjusted as necessary according to factors such as the length of theprobe.

Due to the known degeneracy of the genetic code wherein more than onecodon can encode the same amino acid, a DNA sequence may vary from thatshown in SEQ ID NO:1 and still encode an ADMP having the amino acidsequence of SEQ ID NO:2 or a DNA sequence may vary from that shown inSEQ ID NO:14 and still encode an ADMP having the amino acid sequence ofSEQ ID NO:15. Such variant DNA sequences may result from silentmutations (e.g., occurring during PCR amplification), or may be theproduct of deliberate mutagenesis of a native sequence.

The invention thus provides equivalent isolated DNA sequences encodingbiologically active ADMPs, selected from: (a) the coding region of anative ADMP gene, (b) cDNA comprising the nucleotide sequence presentedin SEQ ID NO:1 or comprising the nucleotide sequence presented in SEQ IDNO:14, (c) DNA capable of hybridization to a DNA of (a) or (b) undermoderately stringent conditions and which encodes a biologically activeADMP, and (d) DNA which is degenerate as a result of the genetic code toa DNA defined in (a), (b) or (c) and which encodes a biologically activeADMP. ADMPs encoded by such DNA equivalent sequences are encompassed bythe invention.

DNAs that are equivalents to the DNA sequence of SEQ ID NO:1 willhybridize under moderately stringent or highly stringent conditions tothe double-stranded native DNA sequence that encode polypeptidescomprising amino acid sequences of 1 to Xaa of SEQ ID NO:2, wherein Xaais an amino acid from 431-837. DNAs that are equivalents to the DNAsequence of SEQ ID NO:14 will hybridize under moderately stringent orhighly stringent conditions to the double-stranded native DNA sequencethat encode polypeptides comprising amino acid sequences of 1 to Xaa ofSEQ ID NO:15, wherein Xaa is an amino acid from 479-930. Examples ofADMP proteins encoded by such DNA, include, but are not limited to, ADMPfragments and ADMPs comprising inactivated N-glycosylation site(s),inactivated KEX2 protease processing site(s), or conservative amino acidsubstitution(s), as described above. ADMP proteins encoded by DNAderived from other species, wherein the DNA will hybridize underconditions of moderate or high stringency to the complement of the cDNAof SEQ ID NO:1 or the cDNA of SEQ ID NO:14 are also encompassed by thisinvention.

ADMP polypeptides may exist as oligomers, such as covalently-linked ornon-covalently-linked dimers or trimers. oligomers may be linked bydisulfide bonds formed between cysteine residues on differentaggrecanase polypeptides. In one embodiment of the invention, an ADMPdimer is created by fusing the ADMP to the Fc region of an antibody(e.g., IgGI) in a manner that does not interfere with biologicalactivity of the ADMP. The Fc polypeptide preferably is fused to theC-terminus of a soluble ADMP. General preparation of fusion proteinscomprising heterologous polypeptides fused to various portions ofantibody-derived polypeptides (including the Fc domain) has beendescribed, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991) and Byrnet al. (Nature 344:677, 1990). A gene fusion encoding the ADMP:Fc fusionprotein is inserted into an appropriate expression vector. ADMP:Fcfusion proteins are allowed to assemble much like antibody molecules,whereupon interchain disulfide bonds form between Fc polypeptides,yielding a divalent ADMP. If fusion proteins are made with both heavyand light chains of an antibody, it is possible to form an aggrecanaseoligomer with as many as four aggrecanase molecules. Alternatively, onecan link two soluble aggrecanase domains with a peptide linker.

Expression vectors containing a nucleic acid sequence encoding an ADMPcan be utilized to produce recombinant protein. An ADMP DNA sequence canbe operably linked to suitable transcriptional and translationalregulatory nucleotide sequences using established procedures. Regulatorysequences, which are usually derived from viral, mammalian or insectgenes, can include transcriptional promoters, operators, or enhancers,mRNA ribosomal binding sites, and/or other appropriate sequences whichdrive transcription, translation initiation and termination. When aregulatory sequence is functionally related to the ADMP DNA sequence,the nucleotide sequence is operably linked. Thus, a promoter nucleotidesequence is operably linked to an ADMP DNA sequence if the promoternucleotide sequence drives the transcription of the ADMP DNA sequence.The expression vector may additionally include an origin of replication,to mediate replication in the desired host cells, as well as aselectable marker gene for the identification and selection oftransformants or transfectants.

Expression vectors may also include signal peptide sequences (secretoryleaders), which may be fused in-frame to the ADMP sequence. Theinclusion of the signal sequence on the resultant fusion protein canenhance extracellular secretion of the ADMP polypeptide. The signalpeptide may be cleaved from the ADMP protein upon export through thecellular secretory pathway.

Host cells for expression of ADMP proteins include prokaryotes and yeastor higher eukaryotic cells. Appropriate cloning and expression vectorsfor use with fungal, yeast, and mammalian cellular hosts are describedfor example, in Pouwels et al. Cloning Vectors: A Laboratory Manual,Elsevier, N.Y., (1985). In vitro translation systems could also beemployed to produce ADMP polypeptides using RNAs derived from DNAconstructs disclosed herein.

Prokaryotes include gram negative or gram positive organisms, forexample, E. coli or Bacilli. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic hostcell, such as E. coli, an ADMP polypeptide may include an N-terminalmethionine residue to facilitate expression of the recombinantpolypeptide in the prokaryotic host cell. The N-terminal Met may becleaved from the expressed recombinant ADMP polypeptide.

Prokaryotic expression vectors generally comprise one or more phenotypicselectable marker genes. Examples of phenotypic selectable marker genesinclude a gene encoding a protein that confers antibiotic resistance(amplicillin, tetracycline, kanamycin), or that supplies an autotrophicrequirement (leucine). Examples of useful expression vectors forprokaryotic host cells include those derived from commercially availableplasmids such as the cloning vector pBR322 (ATCC 37017). The cloningvector pBR322 contains genes for ampicillin and tetracycline resistanceand thus provides simple means for identifying transformed cells. Toconstruct an expression vector using pBR322, an appropriate promoter andan ADMP DNA sequence are inserted into the pBR322 vector. Othercommercially available vectors include for example, pET (Novagen,Madison, Wis., USA) and PGEMI (Promega Biotec, Madison, Wis., USA).

Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include B-lactamase (penicillinase), lactose promotersystem (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature281:544, 1979), a phage l P_(L) promoter, tryptophan (trp) promotersystem (Goeddel et al., Nucl. Acids Res. 8:4057, 1980) and tac promoter(Maniatis, Molecular Cloning: A Laboratory Mammal, Cold Spring HarborLaboratory, p. 412, 1982).

ADMP polypeptides alternatively may be expressed in yeast host cells,preferably from the Saccharomyces genus (e.g., S. cerevisiae). Othergenera of yeast, such as Pichia K.lactis or Kluyveromyces, may also beemployed. Yeast vectors will often contain an origin of replicationsequence from a 2 u yeast plasmid, an autonomously replicating sequence(ARS), a promoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include among others, promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7,149, 1968; and Holland et al., Blochem. L7:4900, 1978),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Other appropriatevectors and promoters for use in yeast expression are described inHiitzeman, EPA-73,657 or in Fleer et al., Gene, 107:285-195 (1991); andvan den Berg et. al., Bio/Technology, 8:135-139 (1990). Anotheralternative is the glucoserepressible ADH2 promoter described by Russellet al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature 300:724,1982). Shuttle vectors replicable in both yeast and E. coli may beconstructed by inserting DNA sequences from pBR322 for selection andreplication in E. coli (Ampr gene and origin of replication) into theabove-described yeast vectors.

The yeast a-factor leader sequence, typically inserted between thepromoter and the cDNA to be expressed, may be employed to mediatesecretion of an ADMP polypeptide. See, e.g., Kurjan et al., Cell 3D:933,1982; Bitter et al., and Proc. Natl. Acad. SCI. USA 11:53301, 1984.Other leader sequences suitable for facilitating secretion ofrecombinant polypeptides from yeast hosts are known to those skilled inthe art. A leader sequence may be modified near its 3′ end to containone or more restriction sites. This will facilitate fusion of the leadersequence to the structural gene.

Yeast transformation protocols are known to those skilled in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 11:1929, 1978. The Hinnen et al. protocol selects forTrp+transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 ug/ml adenine and 20 ug/ml uracil.

Yeast host cells transformed by vectors containing ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 ug/ml adenine and 80 ug/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

The expression of recombinant ADMP polypeptides can also be carried outin mammalian or insect host cell culture systems. Established cell linesof mammalian origin may also be employed. Examples of suitable mammalianhost cell lines include L cells, C127 cells, 3T3 cells (ATCC CCL 163),the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al.,Cell 2.1:175, 1981), HeLa cells, Chinese hamster ovary (CHO) cells, andBHK (ATCC CRL 10) cell lines, and the CV-l/EBNA-1 cell line derived fromthe African green monkey kidney cell line CVI (ATCC CCL 70) as describedby McMahan et al. (EMBO J. 10: 2821, 1991). Baculovirus systems forproduction of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 20 (1988).

Generally, the expression of eukaryotic genes in mammalian host cells isdriven by viral-genome-derived early and late promoters, enhancer,splice signals and polyadenylation sites, which are included in avariety of mammalian expression vectors. Viral early and late promotersare particularly useful because both are easily obtained from a viralgenome as a fragment which may also contain a viral origin ofreplication (Fiers et al.. Nature 273: 113, 1978). Typically used viraltranscriptional and translational control sequences are derived fromRous sarcoma virus, Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40),and human cytomegalovirus (CMV).

An isolated and purified ADMP protein according to the invention may beproduced by recombinant expression systems as described above orpurified from media of stimulated tissue or cells. ADMPs can besubstantially purified, as indicated by a single protein band uponanalysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Oneprocess for producing an ADMP consists of culturing a host celltransformed with an expression vector containing a DNA sequence thatencodes an ADMP under conditions sufficient to promote expression of theADMP. The ADMP is then recovered from culture medium or cell extracts,depending on the expression system used. As known to one skilled in theart, procedures for purifying a recombinant protein will vary accordingto such factors as the type of host cells used and whether or not therecombinant protein is secreted into the culture medium. For example,when expression systems that secrete the recombinant protein areemployed, the culture medium first may be concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore, Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium, or an anion or a cation exchangeresin of the type commonly used in protein purification. Finally, one ormore reverse-phase high-performance liquid chromatography (RP-HPLC)steps utilizing hydrophobic RP-HPLC media can be employed to furtherpurify the ADMP. Some or all of the foregoing purification steps, invarious combinations, can be used to provide an isolated and purifiedrecombinant protein.

In addition to recombinantly producing ADMPs, ADMPs may be isolated andpurified from conditioned media of stimulated bovine nasal cartilagecultures, such stimulation effected with cytokines such as IL-1 or TNF,retinoic acid, adhesion molecule fragments such as fibronectin fragmentsor other stimuli. Other sources of aggrecanase may be used, includingbut not limited to, cartilage and other aggrecanase-expressing tissuesfrom various species, and ADMPs may also be produced by stimulated cellsin culture. ADMP probes containing nucleic acid sequences that hybridizeto native ADMP nucleotide sequence, available through the invention, canbe used to enable identification of cell lines, cells or tissue sourcesof ADMPs. Once a source of ADMPs is identified, ADMPs may be isolatedand purified by optimally stimulating the source cells or tissue toproduce ADMPs.

It is possible to utilize an affinity column comprising an ADMP-bindingprotein to affinity-purify expressed ADMP polypeptides. ADMPpolypeptides can be removed from an affinity column using conventionaltechniques, e.g., in a high salt elution buffer and then dialyzed into alower salt buffer for use or by changing pH or other componentsdepending on the affinity matrix utilized. Example 5 describes aprocedure for employing ADMPs of the invention to generate antibodiesdirected against the ADMPs.

Recombinant protein produced in bacterial culture is usually isolated byinitial disruption of the host cells, centrifugation, extraction fromcell pellets if an insoluble polypeptide, or from the supernatant fluidif a soluble polypeptide. This isolation is followed by one or moreconcentrating steps such as salting-out, ion exchange, affinitypurification or size exclusion chromatography. RP-HPLC can be employedfor final purification steps. Microbial cells can be disrupted by anyconvenient method including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell-lysing agents.

Transformed yeast host cells are preferably used to express an ADMP as asecreted polypeptide in order to simplify purification. Secretedrecombinant polypeptide from a yeast host cell fermentation can bepurified by methods analogous to those disclosed by Urdal et al. (J.Chromatog. 2k:171, 1984).

Antisense or sense oligonucleotides comprising a single-stranded nucleicacid sequence (either RNA or DNA) capable of binding to a target ADMPmRNA sequence (forming a duplex) or to the ADMP sequence in thedouble-stranded DNA helix (forming a triple helix) can be made accordingto the invention. Antisense or sense oligonucleotides, according to thepresent invention, comprise a fragment of the coding region of an ADMPcDNA. Such a fragment generally comprises at least about 14 nucleotides,preferably from about 14 to about 30 nucleotides. The ability to createan antisense or a sense oligonucleotide, based upon a cDNA sequence fora given protein is described in, for example, Stein and Cohen, CancerRes. 48:2659, 1988 and van der Krol et al., BioTechniques 6:958, 1988.

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of complexes that block translation(RNA), or transcription (DNA) by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation. Thus, the antisense oligonucleotides maybe used to block expression of ADMP proteins. Antisense or senseoligonucleotides further comprise oligonucleotides having modifiedsugar-phosphodiester backbones (or other sugar linkages) and whereinsuch sugar linkages are resistant to endbgenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences. Otherexamples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties andother moieties that increases affinity of the oligonucleotide for atarget nucleic acid sequence, such as poly-(L-lysine). Further still,intercalating agents and alkylating agents or metal complexes may beattached to sense or antisense oligonucleotides to modify bindingspecificities of the antisense or sense oligonucleotide for the targetnucleotide sequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO4-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. Antisense or sense oligonucleotidcs are preferably introducedinto a cell containing the target nucleic acid sequence by insertion ofthe antisense or sense oligonucleotide into a suitable retroviralvector, then contacting the cell with the retrovirus vector containingthe inserted sequence, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, the murine retrovirus M-MuLV,N2 (a retrovirus derived from M-MuLV), or the double copy vectorsdesignated DCT5A, DCT5B and DCT5C.

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the nucleotide sequence by formation of a conjugate with aligand binding molecule. Suitable ligand binding molecules include, butare not limited to, cell surface receptors, growth factors, othercytokines, or other ligands that bind to cell surface receptors.Preferably, conjugation of the ligand binding molecule does notsubstantially interfere with the ability of the ligand binding moleculeto bind to its corresponding molecule or receptor, or block entry of thesense or antisense oligonucleotide or its conjugated version into thecell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex. The sense or antisenseoligonucleotide-lipid complex is preferably dissociated within the cellby an endogenous lipase.

Detection of ADMP enzymatic activity in crude culture media from tissueor cell cultures or partially-purified or purified ADMP preparations canbe achieved by incubating the ADMP-containing material with an aggrecansubstrate and monitoring the production of aggrecan fragments generatedby specific cleavage at the Glu373-Ala374 bond using a neoepitopeantibody to either the new N-terminus, ARGSV, or the new C-terminus,NITEGE, formed by cleavage at this bond. The ARGSV neoepitope antibodiesused encompass, but are not limited to, the BC-3 monoclonal antibody(Hughes, C. E., et al., Biochem. J. 306:799-804, 1995). As used herein,“aggrecan” refers to the aggregating proteoglycan, aggrecan, from humanor animal cartilage, as the native aggrecan isolated from tissue, asrecobinant full-length aggrecan or as a recombinant protein representinga portion of the aggrecan molecule. Within an aspect of the invention,an ADMP may be utilized to identify additional ADMP-sensitive sites thusenabling activity to also be detected by monitoring the production offragments formed by cleavage at alternative ADMP-sensitive sites usingneoepitope antibodies to the new C-terminus or to the new N-terminusgenerated by ADMP-specific cleavage at these sites. Alternative sites inthe aggrecan core protein encompass, but are not limited to theE1545-G1546, E1714-G1715, E1819-A1820, or E1919-L1920 bond (numberingbased on the human aggrecan-core protein sequence). These human aggrecanADMP-senstitive cleavage sites are conserved in aggrecan from variousanimal species although the absolute numbering based on the sequence ofthe aggrecan core protein may vary from species to species. Conservedamino acid sequences in various species around conserved ADMP-sensitivesites are shown below.

Human NITEGE³⁷³ ³⁷⁴ARGSVILT Bovine NITEGE ARGSVILT Rat NITEGE ARGNVILTMouse NVTEGE ALGSVILT Pig NITEGE ARGTVILT Sheep NITEGE ARGNVILT ChickenNVTEEE ARGSI Horse NITEGE ARGNVILT Human ASTASELE¹⁵⁴⁵ ¹⁵⁴⁶GRGTIGISBovine ATTAGELE GRGTIDIS Mouse ATTSSELE GRGTIGIS Rat ATTASELE GRGTISVSHuman PTTFKEEE¹⁷¹⁴ ¹⁷¹⁵GLGSVELS Bovine PTTFKEEE GLGSVELS Rat PTTFREEEGLGSVELS Mouse PTTFREEE GLGSVELS Human TQAPTAQE¹⁸¹⁹ ¹⁸²⁰AGEGPSGI BovineTQAPTAQE AGEGPSGI Rat TLAPTAQE AGEGPSSI Mouse TQAPTAQE AGEGPSGI ChickenTQTSVAQE VGEGPSGM Human TEPTISQE¹⁹¹⁹ ¹⁹²⁰LGQRPPVT Bovine TEPTVSQELGQRPPVT Rat TEPTVSQE LGHGPSMT Mouse TEPTVSQE LGHGPSMT Chicken TRPTVSQELGGETAVT Dog TEPTVSQE LAQRPPVT

Thus, aggrecan from various animal species, including but not limitedto, bovine, dog, pig, rat, mouse, sheep, horse and chicken may also beused as a substrate for detecting ADMP activity. Utilizing neoepitopeantibodies allows detection of fragments formed specifically byADMP-mediated cleavage even in the presence of other proteolyticactivities that may be present in crude preparations.

As used herein, the cleavage site “E373-374A” refers to theITEGE373-374ARGS bond of human aggrecan as well as to the homologousaggrecanase-sensitive cleavage site in aggrecan from various animalspecies, the cleavage site “E1545-1546G” refers to the SELE1545-1546GRGTbond of human aggrecan as well as to the homologousaggrecanase-sensitive cleavage site in aggrecan from various animalspecies, the cleavage site “E1714-1715G” refers to the KEEE1714-1715GLGSbond of human aggrecan as well as to the homologousaggrecanase-sensitive cleavage site in aggrecan from various animalspecies, the cleavage site “E1819-1820A” refers to the TAQE1819-1820AGEGbond of human aggrecan as well as to the homologousaggrecanase-sensitive cleavage site in aggrecan from various animalspecies, the cleavage site “E1919-1920L” refers to the ISQE1919-1920LGQRbond of human aggrecan as well as to the homologousaggrecanase-sensitive cleavage site in aggrecan from various animalspecies.

A purified ADMP may also be assayed using any of a variety of proteaseassays known in the art. In general, an ADMP can be assayed through theuse of a peptide substrate that represents the natural cleavage site ofaggrecan cleavage. For example, in order to detect the cleavage of asubstrate by an ADMP, the substrate can be tagged with a fluorescentgroup on one side of the cleavage site and with a fluorescence-quenchinggroup on the opposite side of the cleavage site. Upon cleavage by theADMP, quenching is eliminated thus providing a detectable signal.Alternatively, the substrate may be tagged with a calorimetric leavinggroup that more strongly absorbs upon cleavage. Alternatively, thesubstrate may have a thioester group synthesized into the cleavage siteof the substrate so that upon cleavage by an ADMP, the thiol groupremains and can be easily detected using conventional methods.

Within an aspect of the invention, an ADMP and peptides based on theamino acid sequence of the ADMP, may be utilized to prepare antibodiesthat specifically bind to the ADMP. Specific examples of such antibodypreparation is described in Example 5 and 6 herein. The term“antibodies” is meant to include polyconal antibodies, monoclonalantibodies, fragments thereof such as F(ab′)2, and Fab, as well as anyrecombinantly produced binding parameters. Antibodies are defined to bespecifically binding if they bind an ADMP with a Ka of greater than orequal to about 1×10⁻⁷ M. Affinities of binding partners or antibodiescan be readily determined using conventional techniques, for examplethose described by Scatchard et al., Ann. N.Y. Acad. Sci., 51:660(1949).

Using standard procedures, polyclonal antibodies can be readilygenerated from a variety of sources such as horses, cows, goats, sheep,dogs, chickens, rabbits, mice or rats. In general a purified ADMP, or apeptide based on the amino acid sequence of the ADMP, that isappropriately conjugated is administered to the host animal typicallythrough parenteral injection. The immunogenicity of the ADMP may beenhanced by the use of an adjuvant such as Freund's complete orincomplete adjuvant. Following booster immunizations, small samples ofserum are collected and tested for reactivity to the ADMP or the ADMPpeptides. Examples of various assays useful for such determinationinclude those described in: Antibodies: A Laboratory Manual, Harlow andLane (eds.), Cold Spring Harbor Laboratory Press, 1988; as well asprocedures such as countercurrent immuno-electrophoresis (CIEP),radioimmunoassay radio-immunoprecipitation, enzyme-linked immuno-sorbentassays (ELISA), dot-blot assays, and sandwich assays.

Monoclonal antibodies can be readily prepared using standard proceduressuch as those described in Monoclonal Antibodies, Hybridomas: A NewDimension in Biological Analyses, Plenum Press, Kennett, McKearn, andBechtol (eds.), 1980. The host animals, for example mice, are injectedintraperitoneally at least once, and preferably at least twice atapproximate 3 week intervals, with an isolated and purified ADMP orconjugated ADMP peptide, optionally in the presence of adjuvant. Mousesera are then assayed by the conventional dot-blot technique orantibody-capture technique, to determine which animal is best to use inthe production of hybrid cells. Approximately two to three weeks later,the mice are given an intravenous boost of the ADMP or conjugated ADMPpeptide. Mice are subsequently sacrificed and using establishedprotocols, the spleen cells are fused with commercially availablemyeloma cells. The fusing agent can be any suitable agent used in theart, for example, polyethylene glycol. The fused cells are spread ontoplates containing media that allows for their selective growth. Thefused cells are grown for approximately eight days. Supernatant from theresulting hybridomas is collected and added to a plate that has beencoated with goat anti-mouse Ig. Following washes, a label, such asI¹²⁵-ADMP, is added to each well and followed by incubation. Positivewells are subsequently detected by autoradiography. Positive clones canbe grown in bulk culture and the supernatant subsequently purifiedutilizing a Protein A column.

Other types of “antibodies” may be produced using the informationprovided herein in conjunction with the state of knowledge in the art.Humanized antibodies that are capable of specifically binding ADMPs arealso encompassed by the instant invention.

Once isolated and purified, the antibodies against ADMPs can be used todetect the presence of ADMPs in a sample using established assayprotocols. The antibodies of the invention can also be usedtherapeutically to bind to an ADMP and inhibit its activity in vivo.

The purified ADMPs according to the invention will facilitate thediscovery of inhibitors of aggrecanases, and thus, inhibitors ofcartilage aggrecan degradation. The use of a purified ADMP polypeptidein the screening of potential inhibitors thereof is important and canvirtually eliminate the possibility of interfering reactions withcontaminants. Such a screening assay for detecting theaggrecanase-inhibiting activity of a molecule would typically involvemixing the potential inhibitor molecule with an appropriate substrate,incubating an ADMP that is at least substantially purified with themixture, and determining the extent of substrate cleavage. While variousappropriate substrates may be designed for use in the assay, preferablythe native aggrecan monomer or a peptidyl substrate which encompassesthe E³⁷⁴-³⁷⁴A cleavage site within the interglobular domain of theaggrecan core protein.

Alternatively, monitoring cleavage at aggrecanase-sensitive sites withinthe C-terminus of the aggrecan core protein, including E¹⁵⁴⁵-¹⁵⁴⁶G,E¹⁷¹⁴-¹⁷¹⁵G, E¹⁸¹⁹-¹⁸²⁰A, E¹⁹¹⁹-¹⁹²⁰L (numbering based on humanaggrecan-core protein), can be used for detecting aggrecanase-inhibitingactivity of a molecule by employing appropriate peptidyl substrates orthe native aggrecan monomer and neoepitope antibodies.

In addition, ADMP polypeptides can be used for structure-based design ofaggrecanase-inhibitors. Such structure-based design is also known as“rational drug design.” The ADMP polypeptides can be three-dimensionallyanalyzed by X-ray crystallography, nuclear magnetic resonance orhomology modeling. The use of ADMP structural information in molecularmodeling software systems to assist in the inhibitor design andinhibitor-ADMP interaction is also encompassed by the instant invention.Such computer-assisted modeling and drug design can utilize informationsuch as chemical conformational analysis, electrostatic potential of themolecules, and protein folding. A particular method of the inventioncomprises analyzing the three-dimensional structure of ADMPs for likelybinding sites of substrates, synthesizing a new molecule incorporating apredictive reactive site, and assaying the new molecule as describedabove.

The following examples provide an illustration of embodiments of theinvention and should not be construed to limit the scope of theinvention which is set forth in the appended claims. In the followingexamples, all methods described are conventional unless otherwisespecified.

EXAMPLE 1 Purification of ADMP-1

This example describes a method for purifying ADMP-1. ADMP-1 wasisolated and purified from the conditioned media of stimulated bovinenasal cartilage. Thirty liters of conditioned media, from approximately1000 grams of bovine nasal cartilage, was generated by stimulating withinterleukin-1β (IL-1). In order to accumulate ADMPs in culture media,cartilage matrix was first degraded and depleted of endogenous aggrecanby stimulation with 500 ng/ml human recombinant IL-1 for 6 days withmedia changes every 2 days. Cartilage was then stimulated for anadditional 10 days with 500 ng/ml IL-1 to generate accumulation ofsoluble, active ADMPs in the media. By replacing the media andrestimulating the cartilage with IL-1 every other day during theaccumulation phase, approximately 5 times more aggrecanase activity wasgenerated than by allowing accumulation of ADMPs in conditioned mediawithout media change. Media, containing the ADMPs, was frozen at −70° C.for subsequent purification.

All purification steps were performed at 4° C. unless otherwisespecified. Five liters of frozen conditioned media was thawed overnightand supplemented with 1 μM leupeptin, 1 μM pepstatin, 1 mM PMSF (PMSF isphenylmethylsulfonyl-fluoride), and 0.05% Brij-35. This was clarified bypassage through a 1.2 micron Gelman Capsule filter, and loaded onto a20×10 cm Macro S support column at a flow rate of 40 ml/min. The columnwas washed with Buffer A (Buffer A contains 50 mM HEPES (HEPES isN-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid), pH 7.5, 10 mMCaCl₂, 100 mM NaCl, 0.05% (v/v) Brij-35) until the absorbance at 280 nmreturned to the pre-load baseline value. ADMPs were eluted from thecolumn with 750 ml of buffer A containing 1.0 M NaCl.

ADMPs were detected at this point by their ability to cleave purifiedbovine aggrecan monomers isolated from bovine nasal cartilage by thefollowing procedure: Aggrecan was extracted from the cartilage bystirring at 4° C. for 48 hours in 10 volumes of 4M guanidine-HCl in 0.05M sodium acetate, pH 5.8, containing the protease inhibitors, 0.01M EDTA(EDTA is ethylenediaminetetraacetic acid), 0.lM 6-aminohexanoic acid, 2mM PMSF and 0.05M benzamidine HCl. Aggrecan monomers were isolated byequilibrium density gradient centrifugation in cesium chloride [Hascall,V. C. and Sajdera, S. W. (1969) J. Biol. Chem. 244, 2384-2396.] and thebottom of this gradient (d>1.54 g/ml) containing the aggrecan monomers,was dialyzed at 4° C. against water and lyophilized.

These aggrecan monomers (500 nM) were incubated at 37° C. for at least 4hr with ADMPs eluted from the Macro S support column in a final volumeof 200 ul in Buffer B (Buffer B contains 50 mM Tris, pH 7.6, containing0.1 M NaCl and 10 mM CaCl2), quenched with 20 mM EDTA and analyzed foraggrecan fragments produced exclusively by cleavage at the Glu³⁷³-Ala³⁷⁴bond within the aggrecan core protein using the monoclonal antibody,BC-3 (Hughes, C. E., et al., Biochem. J. 306:799-804, 1995). Thisantibody recognizes aggrecan fragments with the N-terminal sequenceA³⁷⁴RGSVIL . . . , generated upon cleavage by ADMPs. The BC-3 antibodyrecognizes this neoepitope only when it is the N-terminus and not whenit is present internally within aggrecan fragments or within the intactaggrecan core protein. Other proteases produced by cartilage in responseto stimulation of chondrocytes do not cleave at the Glu³⁷³-Ala³⁷⁴ site,therefore only products produced upon cleavage by ADMPs are detected.

Removal of the glycosaminoglycan (GAG) side chains from aggrecan isnecessary for the BC-3 antibody to recognize the ARGSVIL epitope of thecore protein. Therefore, to remove GAGs from the aggrecan, samples werewere enzymatically deglycosylated with chondroitinase ABC (#EC4.2.2.4;Seikaguku Co., Kogyo, Japan) 0.1 units/10 ug GAG in Buffer D (Buffer Dcontains 50 mM sodium acetate, pH 6.5, 100 mM NaCl)for 2 hr at 37° C.and then with keratanase(#EC3.2.1.103; Seikaguku Co., Kogyo, Japan) (0.1units/10 ug GAG) and keratanase II (Seikaguku Co., Kogyo, Japan) (0.002units/10 ug GAG) in Buffer D for 2 hr at 37° C.

After digestion, the samples were precipitated with 5 volumes of acetoneand reconstituted in an appropriate volume of SDS-PAGE sample buffer,loaded on 4-12% gradient gels and then separated by SDS-PAGE under nonreducing conditions, transferred overnight to nitrocellulose andimmunolocated with 1:1000 dilution of the monoclonal antibody BC-3.Subsequently, membranes were incubated with goat anti-mouse IgG alkalinephosphatase conjugate and aggrecan catabolites visualized by incubationwith the appropriate substrate (#S3721; Promega Western blot alkalinephosphatase system) for 10-30 min to achieve optimal color development.BC-3-reactive aggrecan fragments were then quantified by scanningdensitometry.

The material that eluted from the Macro S support column with 0.1 M NaClhad about a 20-fold higher specific activity than the starting material.The eluted material was supplemented with Compound A(N3-methyl-(3R)-2-[(2S)-2-[(1R)-2-(hydroxyamino)-1-methyl-2-oxoethyl]-4-methylpentanoyl]hexahydro-3-pyridazinecarboxamide),a hydroxamic acid-based broad spectrum inhibitor of matrixmetalloproteinases that is ineffective as an inhibitor of ADMP activity.The sample was then loaded onto a 10×7.5 cm gelatin-agarose column at aflow rate of 0.5 ml/min. Compound A was added to prevent degradation ofthe gelatin column by matrix metalloproteinases present during thispurification step. Material passing through this column contained theADMP activity and was collected and concentrated 6 to 7-fold using anAmicon Diaflo pressure concentrator fitted with a YM-30 membrane.

ADMP activity is inhibited by both tissue inhibitor ofmetalloproteinases-1 (TIMP-1) and by a number of hydroxamic acid-basedinhibitors of matrix metalloproteinases. Therefore TIMP-1 and ahydroxamate inhibitor of aggrecanase activity were used to furtheraffinity purify ADMP. Compound A was included during the affinitypurification to prevent matrix metalloproteinases present in thematerial from binding to the TIMP-1 or to the hydroxamate inhibitor ofADMP.

The concentrated material from the gelatin-agarose column containing theADMP activity was incubated with 1 uM bovine TIMP-1 in the presence of 1uM Compound A in for at least 30 minutes to allow ADMP to bind to theTIMP-1. The TIMP-1 was subsequently complexed by incubating with aTIMP-1 monoclonal antibody at a 1:5000 dilution for at least 30 minutes.The TIMP-1-antibody complex was then applied to a 10 ml protein Acolumn. The column was washed 3× with Buffer E (Buffer E contains 10 mMTris, pH 7.5, 250 mM NaCl, 0.025% Tween20) and the protein was elutedfrom the column with 100 mM glycine/HCl, pH 2.5.

An ADMP-inhibitor hydroxamate affinity resin was produced in thefollowing manner. POROS 20-NH perfusive chromatographic media(Perseptive Biosystems), a highly crosslinked poly(styrenedivinylbenzene) polymer, was mixed overnight with four equivalents ofFmoc-beta-alanine, four equivalents HBTU(O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate), andeight equivalents DIEA (N,N diisopropylethylamine) in DMF(dimethylformamide). The resin was rinsed several times with DMF, thenthe FMOC group was removed with 20% piperidine/DMF. Thiscoupling/deprotecting scheme was repeated three more times, resulting infour beta-alanine residues coupled to the resin. After the finalpiperidine deprotection, 1.5 equivalents of a t-butyl protectedhydroxamic acid capable of inhibiting ADMP activity was coupled with 1.5equivalents HBTU and 3 equivalents DIEA in DMF. After rinsing with DMFand CH₂Cl₂, the t-butyl group was removed by mixing overnight with TFA(trifluoroacetic acid), leaving the hydroxamic acid. The resin wasthoroughly rinsed with CH₂Cl₂ and dried under vacuum.

The eluate from the protein A column was neutralized with 1 M Tris baseto pH 7.5 and then incubated for 2 hours with the aggrecanase-inhibitorhydroxamate resin at a ratio of 1 mg resin for every 1 ml of eluate.Following the incubation, the resin was spun down and washed at 4° C.with Buffer E, three times, 10 minutes each wash. Bound ADMP was elutedfrom the resin by mixing with approximately 0.5-1.0 ml 4 M GuHCl for 30minutes at room temperature. The eluate, containing the ADMP activity,was dialyzed against Buffer F (Buffer F comprises 50 mM Tris, pH 7.6,100 mM NaCl, 5 mM CaCl₂) for 48 hours at 4° C. A portion of the eluatewas run on a SDS-PAGE gel (10-20%), and silver staining revealed onepredominant protein, ADMP-1, that ran as a doublet between the 64 and 92kDa markers on the gel at approximately 67 kDa.

Incubation of the GuHCl-eluted ADMP-1 with isolated bovine aggrecanproduced a pattern of BC-3-reactive fragments similar to that producedupon cleavage of cartilage aggrecan by endogenous aggrecanase in bovinenasal cartilage stimulated with IL-1.

Binding of this 67 kDa ADMP-1 doublet to the aggrecanase-inhibitorhydroxamate resin was blocked by inclusion during affinity purificationof 10 uM Compound B((2S,11S,12S)-12-isobutyl-2-[(methylamino)carbonyl]-11[(hydroxylamino)carbonyl]-8,13-dioxo-1,7-diazacyclotridecane),a potent ADMP inhibitor. Thus, the binding of the 67 kDa ADMP-1 to theaffinity resin was not affected by the presence of Compound A (a potentinhibitor of matrix metalloproteinases that is inactive in inhibitingADMPs), but was blocked by Compound B (a potent ADMP inhibitor).

A region of the SDS-PAGE gel containing the 67 kDa ADMP-1 protein wasexcised along with a control region of the same gel which did notcontain any detectable protein. Gel slices were incubated with 1% TritonX-100 for 1 hour at room temperature to remove the SDS for the gel. Thegel was then crushed in 1 ml final volume of Buffer G (Buffer G contains50 mM Tris, pH 7.5, 100 mM NaCl, 10 mM CaCl₂) and dialyzed in 10 kDacutoff dialysis membrane for 48 hours against 12 liters of Buffer B at4° C. ADMP activity was then determined by incubation with aggrecansubstrate and monitoring products using the BC-3 antibody. The region ofthe gel containing the 67 kDa ADMP-1 protein exhibited activity whilethat of the control portion of the gel did not.

Generation of Peptides and Sequencing

The two protein bands running as a doublet at approximately 67 kDa andidentified with ADMP activity by elution from SDS gels, were immobilizedon PVDF and subjected to N-terminal amino acid sequence analysis asfollows. The N-terminal sequence analyses were performed using theHewlett-Packard G1005A N-terminal sequencer [Miller, C. G. (1994)Methods: A Companion to Methods in Enzymology 6, 315-333]. Theseanalyses of PVDF-bloted protein were carried out using a modification ofthe Hewlett-Packard Routine 3.0 Sequencing Methods [Hewlettt-Packardtechnical note TNF95-1: Routine 3.0 Sequencing Methods] in combinationwith the Hewlett-Packard PTH 4.M HPLC Method [Hewlett-Packard technicalnote TN95-6: PTH 4.M HPLC Method]. The modifications of the sequencingmethods involved: (1) the replacement of the lower SAX sample columnwith a lower RP adapter column and (2) the substitution of a mixture ofLC-A buffer and water (LC-A:water; 2:1) for solvent S4 at the step wherethe PTH-amino acids were resuspended for injection into the HPLC. Allsequencer reagents were from Hewlett-Packard and the HPLC-grade waterused to dilute the LC-A as described above was from Aldrich ChemicalCompany.

Both bands of the 67 kDa doublet represented the same protein and thesequence of the first 27 residues were determined to be:

SEQ ID NO:4 FASLSRFVETLVVADDKMAAFHGAGLK

Two internal fragments, a 7-mer and a 11-mer, from a tryptic digestionof the 67 kDa ADMP-1 protein were sequenced and have the fo llowingsequences:

SEQ ID NO:5 YTGVAPR

SEQ ID NO:6 ALGYYYVLDP

EXAMPLE 2 Purification of ADMP-2

This example describes a method for purifying a second member of theADMP family, ADMP-2. The concentrated material from the gelatin-agarosecolumn containing the ADMP activity prepared as detailed in Example 1was loaded onto a 3×20 cm phenyl-sepharose column that had beenequilibrated with buffer A containing 10% (w/v) ammonium sulfate andwithout the Brij-35 detergent. The column was then washed with 150 ml ofequilibration buffer. Proteins were eluted with a 400 ml gradient from10% to 0% ammonium sulfate in equilibration buffer at a flow rate of 2ml/min. At the end of the gradient phase, proteins were further elutedfrom the column with an additional 200 ml of 0% ammonium sulfateequilibration buffer. Fractions were collected throughout the loading,washing and elution phases and analyzed for ADMP activity. Fractionscontaining ADMP activity were pooled.

Material that eluted from the phenyl sepharose column with ammoniumsulfate had about 87-fold higher specific activity than the startingmaterial. These pooled fractions were loaded onto a 4×60 cm CM columnthat had been equilibrated with buffer H (buffer H contains 50 mM HEPES,0.1 M NaCl, pH 7.5). The column was washed with 150 ml of buffer H, andthen proteins were eluted from the column with a 300 ml gradient from0.1 to 1.0 M NaCl in buffer H at a flow rate of 1.5 ml/min. Fractionswere collected throughout the loading, washing, and elution phases andanalyzed for ADMP activity. Fractions with high enzymatic activity werepooled. While still containing contaminating proteins, the enzyme atthis stage was of adequate purity for use in high throughput screensdesigned to find small molecular weight inhibitors of ADMP activity.

The pooled fractions from the CM column containing ADMP activity hadabout a 500 fold higher specific activity than the starting material.This material was concentrated approximately 10-fold with an AmiconDiaflo pressure concentrated fitted with a YM-30 membrane. Samples of 2ml of the concentrate were then applied to a 2×200 cm Sephacryl S-200column equilibrated with Buffer H. The column was eluted isocraticallyin the same buffer at a flow rate of 0.2 ml/min. Fractions of 4 ml werecollected throughout the elution and analyzed for ADMP activity. Pooledfractions containing ADMP activity had about a 3500-fold higher specificactivity than the starting material.

The ADMP eluted from the sizing column was concentrated as describedabove to a final volume of 1 ml and injected onto a (30 mm×4.6 mm) C₄alkylsilane-derivatized silica column and eluted with a linear gradientfrom 0 to 50% (v/v) acetonitrile in 0.1% aqueous TFA, at a flow rate of1 ml/min. Fractions were collected throughout the elution phase, andimmediately diluted 10-fold with Buffer A. As long as the acetonitrileconcentration was diluted quickly, good recovery of enzymatic activitywas observed from this column. Pooled fractions containing ADMP activityhad about 100,000-fold higher specific activity than the startingmaterial.

Analysis of the HPLC purified ADMP by SDS-PAGE with silver stainingdemonstrated the presence of multiple protein bands ranging in apparentmolecular weights from approximately 90 to 30 kDa. Prominent bands wereobserved in the range of 65 to 40 kDa. To identify which protein band(s)corresponded to the ADMP activity, two samples of the HPLC purified ADMPwere electrophoretically fractionated in adjacent wells of a 10%Tris-glycine polyacrylamide gel under non-reducing conditions. One lanewas stained with silver. The other lane was cut horizontally into 22approximately equal volume slices, each representing a differentmolecular weight range. The individual slices were crushed and soaked in100 μl of 20 mM Tris, 10 mM CaCl₂ 100 mM NaCl, 2.5% Triton-X100. Thesamples were incubated in this buffer at 4° C. overnight to renature theenzyme and elute it from the gel slice into the supernatant solution.The resulting solutions were tested for ADMP activity. ADMP activity wasassociated with four protein bands, centered at ca. 64, 62, 54 and 50kDa.

Generation of Peptides and Sequencing

The four protein bands identified with ADMP activity by elution from SDSgels were immobilized on PVDF and subjected to N-terminal amino acidsequence analysis. The 64 kDa protein yielded 41 cycles of sequencehaving the following composition:

SEQ ID NO:16 SISRARQVEL LLVADASMAR MYGRGLQHYL LTLASIANKLYF

The 62, 54, and 50 kDa protein bands yielded the same N-terminal aminoacid sequence as found in the 64 kDa band.

Based on these results it appeared that the 64, 62, 54 and 50 kDaprotein bands displaying ADMP activity represent different forms of thesame ADMP-2 protein. The difference in molecular weights of these fourbands is most likely the result of differential processing of the fourforms (e.g., differential glycosylation or C-terminal proteolyticprocessing).

EXAMPLE 3 Cloning of Human ADMP-1

This example describes a procedure for isolating a DNA sequence encodinghuman ADMP-1. N-terminal sequence obtained from the 67-kDa protein wasshown to be 62% identical to the murine ADAMTS-1 protein (K. Kuno, etal. J. Biol. Chem. 272:556-562, 1997), suggesting that the 67 kDaprotein was a member of the ADAM family of proteins. As defined hereinand in the reference above, the name “ADAMTS-1” is an abbreviation for Adisintegrin and metalloproteinase with thrombospondin motifs. Uponsubsequent internal peptide sequencing, SEQ ID NO:6 was shown to be 50%identical to ADAMTS-1 and 91% (10/11) identical to sequences encoded bya murine EST 474985 (Accession number AA041973). Based on the highdegree of identity between the latter internal peptide sequence andsequences encoded by EST474985, we proceeded to clone the humansequences representing the human homologue of murine EST 474985.

Two approaches were utilized in the cloning of the human homologue ofmurine EST 474985. In the first, the cDNA from which EST474985 wasobtained was sequenced in its entirety, with the resulting sequencebeing used to search the EST data base for human ESTs with significanthomology. In the second approach, human sequences were amplified usingPCR primers designed from murine EST 474985 (SEQ ID NO:7 and SEQ IDNO:8). Both approaches provided us with sequence from the humanhomologue. A human EST (Accession number D45652) was identified thatcontained sequences having significant homology to the 1.7 kb murinecDNA from which EST 474985 was derived. The level of identity was 80%overall at the nucleotide level and contained sequences from thenon-coding 3′ untranslated region. Utilizing the second approach, wewere able to obtain a 190 bp amplicon from human heart cDNA. SubsequentDNA sequence analysis indicated that the human PCR product was 89%identical at the nucleotide level to the murine EST, with the deducedpeptide sequences being 96% identical.

We were successful at obtaining additional coding sequences utilizingPCR primers designed from the human EST and the 190 bp PCR product. A2-kb clone was obtained using sense and anti-sense primers designed fromthe 190-bp PCR product (SEQ ID NO:9) and the human 3′ EST (SEQ IDNO:10), respectively. Utilizing antisense primers designed from the190-bp PCR product (SEQ ID NO:11 and SEQ ID NO:12), we were able toclone a 2.2-kb 5′ RACE (rapid amplification of cDNA ends) product. Sixclones from the 5′ and 3′ PCRs were sequenced in order to obtain aconsensus sequence for the cDNA. A total 4.2-kb of sequence has beenassembled (SEQ ID NO:1). The assembled cDNA contains a 2511-bp openreading frame encoding 837 amino acids (SEQ ID NO:2) with multiplein-frame stop codons being present upstream of the start methionine. ThecDNA encodes sequences present in the N-terminal peptide sequence (SEQID NO:4), with the cDNA containing all 27 residues seen in theN-terminal sequence of the 67 kDa ADMP-1 protein. The N-terminus of theisolated 67 kDa ADMP-1 begins with amino acid 213 of SEQ ID NO:2,indicating that this protein lacks the propeptide domain of themolecule. The cDNA also encodes sequences present in the internalpeptide sequences (SEQ ID NO:5 and SEQ ID NO:6), with sequences encodedby the cDNA being identical in six of seven positions for the firstpeptide and ten of eleven positions for the second peptide. The deducedprotein sequence has homology to the previously reported ADAMTS-1protein. Like ADAMTS-1, the ADMP-1 cDNA contains a propeptide domain,metalloproteinase domain and disintegrin-like domain. A noteworthydifference between the ADAMTS-1 and sequences encoded by the ADMP-1 cDNAis the presence of a single thrombospondin-domain in the deducedaggrecan degrading metallo protease, in contrast to the threethrombospondin domains seen in ADAMTS-1.

EXAMPLE 4 Cloning of ADMP-2

This example describes a procedure for isolating a DNA sequence encodinghuman ADMP-2. A closely related family member was identified by usingthe deduced ADMP-1 peptide sequence to search the EST data base.Sequences encoded by murine EST 569515 were shown to be 67% identical tosequences encoded by the ADMP-1 cDNA sequence. Further sequence analysisof the murine cDNA from which the EST 569515 was obtained indicated thatit encoded sequences that were 95% (39/41) identical and 100% similar tothe N-terminal sequence of the purified 50/64 kD aggrecanase, ADMP-2.These data indicated that the murine cDNA encodes the ADMP-2. PCRprimers (SEQ ID NO: 17 and SEQ ID NO: 18) were designed from the murinesequence and were used to amplify a product from human heart cDNA, withthe resulting product being 92% identical (150/163) at the nucleotideand 100% identical at the amino acid level to the murine sequences.Sequences present in the human amplicon were used to design a PCR primerfor use in 3′ RACE (SEQ ID NO: 19). However, only a partial 3′ RACEclones was obtained using this approach. In order to obtain additionalsequences, a human liver cDNA library was screened by PCR as described(D. I. Israel (1993) Nuc. Acids Res. 21, 2627-2631) using PCR primersdesigned from the human amplicon and the partial 3′ RACE clone (SEQ IDNO: 19 and SEQ ID NO: 20). Two cDNA clones were obtained from the liverlibrary, with each clone being approximately 5.5 kb is size.

Sequence analysis of the cDNA clones indicated that both cDNAs contain a2793 bp open reading frame (SEQ ID NO: 14) encoding a 930 amino acidprotein (SEQ ID NO: 15). The deduced protein sequence contains sequencesthat are 97.5% (40/41) identical and 100% similar to the bovineN-terminal peptide sequence of ADMP-2. The predicted protein encodes anADMP family member closely related to human ADMP-1 and to the murineADAMTS-1. All three proteins contain metalloproteinase-domains,disintigrin-like domains and thrombospondin motifs. These three familymembers were found to have a variable number ofthrombospondin-submotifs. Murine ADAMTS-1 has been shown to contains twothrombospondin-submotifs (Kuno et al (1997) J. Biol;. Chem 272,556-562), while the ADMP-2 cDNA encodes one thrombospondin-submotif andthe ADMP-1 lacks the thrombospondin-submotifs altogether. Overall, thepro-domains of the three proteins are the least conserved, with thepercent identity ranging from 15% for the ADMP-1 and ADMP-2 to 33% formADAMTS-1 and ADMP-1. Greatest conservation was seen in the catalyticdomains with the percent identity ranging from 48% (ADMP-1 and ADMP-2)to 62% (mADAMTS-1 and ADMP-1)

EXAMPLE 5 Preparation of Antibodies Against ADMP-1

This example describes a method for generating antibodies againstADMP-1. A peptide based on the N-terminus of the purified 67 kDa ADMP-1was synthesized with the following sequence:

SEQ ID NO:13 CASLSRFVETLVVADDK

The peptide was linked to the carrier protein, keyhole limpethemocyanin, and then subsequently used for immunization of a sheep. Thecoupled peptide antigen was suspended in PBS (phosphate-buffered saline)at 1 mg/ml with an equal volume of complete Freund's adjuvant. Thematerial was mixed until it formed an emulsion, and then the materialwas injected at 6-8 subcutaneous sites. A total of 150-200 ug of coupledpeptide was injected into the animal. The sheep was boosted every twoweeks (for a total of five times) and a production bleed was collectedat each time point. The affinity of the antibody was tested both in anELISA and Western assay using the above antigen peptide conjugated toBSA. The polyclonal antiserum was positive for recognizing the BSAcoupled peptide both in the ELISA and Western assays. The polyclonalsera was affinity purified over an antigen peptide (CASLSRFVETLVVADDK)column to capture the high affinity IgG antibodies and remove the lowaffinity antibodies.

EXAMPLE 6 Preparation of Antibodies Against ADMP-2

This example describes a method for generating antibodies againstADMP-2. A peptide based on the N-terminus of the purified 50/64 kDaADMP-2 was synthesized with the following sequence:

SEQ ID NO: 21: SISRARQVELLAhxC-amide

Aminohexanoic acid (Ahx) was added to lengthen the peptide. The peptidewas linked to the carrier protein, keyhole limpet hemocyanin, and thensubsequently used for immunization of two rabbits. Five immunizationsand bleedings yielded 200-250 ml of serum. The affinity of the antibodywas compared with preimmune sera using an enzyme linked immunosorbentassay (ELISA) with BSA-coupled peptide on the solid phase. For all sera,results were expressed as the reciprocal of the serum dilution thatresulted in an OD405 of 0.2 by detection with alkalinephosphatase-anti-rabbit IgG conjugate and NPP dye. The polyclonalantiserum was positive for recognizing the BSA coupled peptide in theELISA and 90% of the aggrecanase activity in a 41-mer peptide-basedenzymatic assay could be immunoprecipited by the antibody. Thepolyclonal sera was affinity purified over an antigen peptide column tocapture the high affinity IgG antibodies and remove the low affinityantibodies. The resulting affinity-purified antibody works well forrecognizing ADMP-2 bands in Western analysis.

21 1 4192 DNA Homo sapiens CDS (406)..(2916) 1 acagacacat atgcacgagagagacagagg aggaaagaga cagagacaaa ggcacagcgg 60 aagaaggcag agacagggcaggcacagaag cggcccagac agagtcctac agagggagag 120 gccagagaag ctgcagaagacacaggcagg gagagacaaa gatccaggaa aggagggctc 180 aggaggagag tttggagaagccagacccct gggcacctct cccaagccca aggactaagt 240 tttctccatt tcctttaacggtcctcagcc cttctgaaaa ctttgcctct gaccttggca 300 ggagtccaag cccccaggctacagagagga gctttccaaa gctagggtgt ggaggacttg 360 gtgccctaga cggcctcagtccctcccagc tgcagtacca gtgcc atg tcc cag aca 417 Met Ser Gln Thr 1 ggctcg cat ccc ggg agg ggc ttg gca ggg cgc tgg ctg tgg gga gcc 465 Gly SerHis Pro Gly Arg Gly Leu Ala Gly Arg Trp Leu Trp Gly Ala 5 10 15 20 caaccc tgc ctc ctg ctc ccc att gtg ccg ctc tcc tgg ctg gtg tgg 513 Gln ProCys Leu Leu Leu Pro Ile Val Pro Leu Ser Trp Leu Val Trp 25 30 35 ctg cttctg cta ctg ctg gcc tct ctc ctg ccc tca gcc cgg ctg gcc 561 Leu Leu LeuLeu Leu Leu Ala Ser Leu Leu Pro Ser Ala Arg Leu Ala 40 45 50 agc ccc ctcccc cgg gag gag gag atc gtg ttt cca gag aag ctc aac 609 Ser Pro Leu ProArg Glu Glu Glu Ile Val Phe Pro Glu Lys Leu Asn 55 60 65 ggc agc gtc ctgcct ggc tcg ggc gcc cct gcc agg ctg ttg tgc cgc 657 Gly Ser Val Leu ProGly Ser Gly Ala Pro Ala Arg Leu Leu Cys Arg 70 75 80 ttg cag gcc ttt ggggag acg ctg cta cta gag ctg gag cag gac tcc 705 Leu Gln Ala Phe Gly GluThr Leu Leu Leu Glu Leu Glu Gln Asp Ser 85 90 95 100 ggt gtg cag gtc gagggg ctg aca gtg cag tac ctg ggc cag gcg cct 753 Gly Val Gln Val Glu GlyLeu Thr Val Gln Tyr Leu Gly Gln Ala Pro 105 110 115 gag ctg ctg ggt ggagca gag cct ggc acc tac ctg act ggc acc atc 801 Glu Leu Leu Gly Gly AlaGlu Pro Gly Thr Tyr Leu Thr Gly Thr Ile 120 125 130 aat gga gat ccg gagtcg gtg gca tct ctg cac tgg gat ggg gga gcc 849 Asn Gly Asp Pro Glu SerVal Ala Ser Leu His Trp Asp Gly Gly Ala 135 140 145 ctg tta ggc gtg ttacaa tat cgg ggg gct gaa ctc cac ctc cag ccc 897 Leu Leu Gly Val Leu GlnTyr Arg Gly Ala Glu Leu His Leu Gln Pro 150 155 160 ctg gag gga ggc acccct aac tct gct ggg gga cct ggg gct cac atc 945 Leu Glu Gly Gly Thr ProAsn Ser Ala Gly Gly Pro Gly Ala His Ile 165 170 175 180 cta cgc cgg aagagt cct gcc agc ggt caa ggt ccc atg tgc aac gtc 993 Leu Arg Arg Lys SerPro Ala Ser Gly Gln Gly Pro Met Cys Asn Val 185 190 195 aag gct cct cttgga agc ccc agc ccc aga ccc cga aga gcc aag cgc 1041 Lys Ala Pro Leu GlySer Pro Ser Pro Arg Pro Arg Arg Ala Lys Arg 200 205 210 ttt gct tca ctgagt aga ttt gtg gag aca ctg gtg gtg gca gat gac 1089 Phe Ala Ser Leu SerArg Phe Val Glu Thr Leu Val Val Ala Asp Asp 215 220 225 aag atg gcc gcattc cac ggt gcg ggg cta aag cgc tac ctg cta aca 1137 Lys Met Ala Ala PheHis Gly Ala Gly Leu Lys Arg Tyr Leu Leu Thr 230 235 240 gtg atg gca gcagca gcc aag gcc ttc aag cac cca agc atc cgc aat 1185 Val Met Ala Ala AlaAla Lys Ala Phe Lys His Pro Ser Ile Arg Asn 245 250 255 260 cct gtc agcttg gtg gtg act cgg cta gtg atc ctg ggg tca ggc gag 1233 Pro Val Ser LeuVal Val Thr Arg Leu Val Ile Leu Gly Ser Gly Glu 265 270 275 gag ggg ccccaa gtg ggg ccc agt gct gcc cag acc ctg cgc agc ttc 1281 Glu Gly Pro GlnVal Gly Pro Ser Ala Ala Gln Thr Leu Arg Ser Phe 280 285 290 tgt gcc tggcag cgg ggc ctc aac acc cct gag gac tcg gac cct gac 1329 Cys Ala Trp GlnArg Gly Leu Asn Thr Pro Glu Asp Ser Asp Pro Asp 295 300 305 cac ttt gacaca gcc att ctg ttt acc cgt cag gac ctg tgt gga gtc 1377 His Phe Asp ThrAla Ile Leu Phe Thr Arg Gln Asp Leu Cys Gly Val 310 315 320 tcc act tgcgac acg ctg ggt atg gct gat gtg ggc acc gtc tgt gac 1425 Ser Thr Cys AspThr Leu Gly Met Ala Asp Val Gly Thr Val Cys Asp 325 330 335 340 ccg gctcgg agc tgt gcc att gtg gag gat gat ggg ctc cag tca gcc 1473 Pro Ala ArgSer Cys Ala Ile Val Glu Asp Asp Gly Leu Gln Ser Ala 345 350 355 ttc actgct gct cat gaa ctg ggt cat gtc ttc aac atg ctc cat gac 1521 Phe Thr AlaAla His Glu Leu Gly His Val Phe Asn Met Leu His Asp 360 365 370 aac tccaag cca tgc atc agt ttg aat ggg cct ttg agc acc tct cgc 1569 Asn Ser LysPro Cys Ile Ser Leu Asn Gly Pro Leu Ser Thr Ser Arg 375 380 385 cat gtcatg gcc cct gtg atg gct cat gtg gat cct gag gag ccc tgg 1617 His Val MetAla Pro Val Met Ala His Val Asp Pro Glu Glu Pro Trp 390 395 400 tcc ccctgc agt gcc cgc ttc atc act gac ttc ctg gac aat ggc tat 1665 Ser Pro CysSer Ala Arg Phe Ile Thr Asp Phe Leu Asp Asn Gly Tyr 405 410 415 420 gggcac tgt ctc tta gac aaa cca gag gct cca ttg cat ctg cct gtg 1713 Gly HisCys Leu Leu Asp Lys Pro Glu Ala Pro Leu His Leu Pro Val 425 430 435 actttc cct ggc aag gac tat gat gct gac cgc cag tgc cag ctg acc 1761 Thr PhePro Gly Lys Asp Tyr Asp Ala Asp Arg Gln Cys Gln Leu Thr 440 445 450 ttcggg ccc gac tca cgc cat tgt cca cag ctg ccg ccg ccc tgt gct 1809 Phe GlyPro Asp Ser Arg His Cys Pro Gln Leu Pro Pro Pro Cys Ala 455 460 465 gccctc tgg tgc tct ggc cac ctc aat ggc cat gcc atg tgc cag acc 1857 Ala LeuTrp Cys Ser Gly His Leu Asn Gly His Ala Met Cys Gln Thr 470 475 480 aaacac tcg ccc tgg gcc gat ggc aca ccc tgc ggg ccc gca cag gcc 1905 Lys HisSer Pro Trp Ala Asp Gly Thr Pro Cys Gly Pro Ala Gln Ala 485 490 495 500tgc atg ggt ggt cgc tgc ctc cac atg gac cag ctc cag gac ttc aat 1953 CysMet Gly Gly Arg Cys Leu His Met Asp Gln Leu Gln Asp Phe Asn 505 510 515att cca cag gct ggt ggc tgg ggt cct tgg gga cca tgg ggt gac tgc 2001 IlePro Gln Ala Gly Gly Trp Gly Pro Trp Gly Pro Trp Gly Asp Cys 520 525 530tct cgg acc tgt ggg ggt ggt gtc cag ttc tcc tcc cga gac tgc acg 2049 SerArg Thr Cys Gly Gly Gly Val Gln Phe Ser Ser Arg Asp Cys Thr 535 540 545agg cct gtc ccc cgg aat ggt ggc aag tac tgt gag ggc cgc cgt acc 2097 ArgPro Val Pro Arg Asn Gly Gly Lys Tyr Cys Glu Gly Arg Arg Thr 550 555 560cgc ttc cgc tcc tgc aac act gag gac tgc cca act ggc tca gcc ctg 2145 ArgPhe Arg Ser Cys Asn Thr Glu Asp Cys Pro Thr Gly Ser Ala Leu 565 570 575580 acc ttc cgc gag gag cag tgt gct gcc tac aac cac cgc acc gac ctc 2193Thr Phe Arg Glu Glu Gln Cys Ala Ala Tyr Asn His Arg Thr Asp Leu 585 590595 ttc aag agc ttc cca ggg ccc atg gac tgg gtt cct cgc tac aca ggc 2241Phe Lys Ser Phe Pro Gly Pro Met Asp Trp Val Pro Arg Tyr Thr Gly 600 605610 gtg gcc ccc cag gac cag tgc aaa ctc acc tgc cag gcc cgg gca ctg 2289Val Ala Pro Gln Asp Gln Cys Lys Leu Thr Cys Gln Ala Arg Ala Leu 615 620625 ggc tac tac tat gtg ctg gag cca cgg gtg gta gat ggg acc ccc tgt 2337Gly Tyr Tyr Tyr Val Leu Glu Pro Arg Val Val Asp Gly Thr Pro Cys 630 635640 tcc ccg gac agc tcc tcg gtc tgt gtc cag ggc cga tgc atc cat gct 2385Ser Pro Asp Ser Ser Ser Val Cys Val Gln Gly Arg Cys Ile His Ala 645 650655 660 ggc tgt gat cgc atc att ggc tcc aag aag aag ttt gac aag tgc atg2433 Gly Cys Asp Arg Ile Ile Gly Ser Lys Lys Lys Phe Asp Lys Cys Met 665670 675 gtg tgc gga ggg gac ggt tct ggt tgc agc aag cag tca ggc tcc ttc2481 Val Cys Gly Gly Asp Gly Ser Gly Cys Ser Lys Gln Ser Gly Ser Phe 680685 690 agg aaa ttc agg tac gga tac aac aat gtg gtc act atc ccc gcg ggg2529 Arg Lys Phe Arg Tyr Gly Tyr Asn Asn Val Val Thr Ile Pro Ala Gly 695700 705 gcc acc cac att ctt gtc cgg cag cag gga aac cct ggc cac cgg agc2577 Ala Thr His Ile Leu Val Arg Gln Gln Gly Asn Pro Gly His Arg Ser 710715 720 atc tac ttg gcc ctg aag ctg cca gat ggc tcc tat gcc ctc aat ggt2625 Ile Tyr Leu Ala Leu Lys Leu Pro Asp Gly Ser Tyr Ala Leu Asn Gly 725730 735 740 gaa tac acg ctg atg ccc tcc ccc aca gat gtg gta ctg cct ggggca 2673 Glu Tyr Thr Leu Met Pro Ser Pro Thr Asp Val Val Leu Pro Gly Ala745 750 755 gtc agc ttg cgc tac agc ggg gcc act gca gcc tca gag aca ctgtca 2721 Val Ser Leu Arg Tyr Ser Gly Ala Thr Ala Ala Ser Glu Thr Leu Ser760 765 770 ggc cat ggg cca ctg gcc cag cct ttg aca ctg caa gtc cta gtggct 2769 Gly His Gly Pro Leu Ala Gln Pro Leu Thr Leu Gln Val Leu Val Ala775 780 785 ggc aac ccc cag gac aca cgc ctc cga tac agc ttc ttc gtg ccccgg 2817 Gly Asn Pro Gln Asp Thr Arg Leu Arg Tyr Ser Phe Phe Val Pro Arg790 795 800 ccg acc cct tca acg cca cgc ccc act ccc cag gac tgg ctg caccga 2865 Pro Thr Pro Ser Thr Pro Arg Pro Thr Pro Gln Asp Trp Leu His Arg805 810 815 820 aga gca cag att ctg gag atc ctt cgg cgg cgc ccc tgg gcgggc agg 2913 Arg Ala Gln Ile Leu Glu Ile Leu Arg Arg Arg Pro Trp Ala GlyArg 825 830 835 aaa taacctcact atcccggctg ccctttctgg gcaccggggcctcggactta 2966 Lys gctgggagaa agagagagct tctgttgctg cctcatgctaagactcagtg gggaggggct 3026 gtgggcgtga gacctgcccc tcctctctgc cctaatgcgcaggctggccc tgccctggtt 3086 tcctgccctg ggaggcagtg atgggttagt ggatggaaggggctgacaga cagccctcca 3146 tctaaactgc cccctctgcc ctgcgggtca caggagggagggggaaggca gggagggcct 3206 gggccccagt tgtatttatt tagtatttat tcacttttatttagcaccag ggaaggggac 3266 aaggactagg gtcctgggga acctgacccc tgacccctcatagccctcac cctggggcta 3326 ggaaatccag ggtggtggtg ataggtataa gtggtgtgtgtatgcgtgtg tgtgtgtgtg 3386 tgaaaatgtg tgtgtgctta tgtatgaggt acaacctgttctgctttcct cttcctgaat 3446 tttatttttt gggaaaagaa aagtcaaggg tagggtgggccttcagggag tgagggatta 3506 tccttttttt tttctttctt tctttctttt tttttttgagacagaatctc gctctgtcgc 3566 ccaggctgga gtgcaatggc acaatctcgg ctcactgcatcctccgcctc ccgggttcaa 3626 gtgattctca tgcctcagcc tcctgagtag ctgggattacaggctcctgc caccacgccc 3686 ggctaatttt tgttttgttt tgtttggaga cagagtctcgctattgtcac cagggctgga 3746 atgatttcag ctcactgcaa ccttcgccac ctgggttccagcaattctcc tgcctcagcc 3806 tcccgagtag ctgagattat aggcacctac caccacgcccggctaatttt tgtattttta 3866 gtagagacgg ggtttcacca tgttggccag gctggtctcgaactcctgac cttaggtgat 3926 ccactcgcct tcatctccca aagtgctggg attacaggcgtgagccaccg tgcctggcca 3986 cgcccaacta atttttgtat ttttagtaga gacagggtttcaccatgttg gccaggctgc 4046 tcttgaactc ctgacctcag gtaatcgacc tgcctcggcctcccaaagtg ctgggattac 4106 aggtgtgagc caccacgccc ggtacatatt ttttaaattgaattctacta tttatgtgat 4166 ccttttggag tcagacagat gtgggt 4192 2 837 PRTHomo sapiens 2 Met Ser Gln Thr Gly Ser His Pro Gly Arg Gly Leu Ala GlyArg Trp 1 5 10 15 Leu Trp Gly Ala Gln Pro Cys Leu Leu Leu Pro Ile ValPro Leu Ser 20 25 30 Trp Leu Val Trp Leu Leu Leu Leu Leu Leu Ala Ser LeuLeu Pro Ser 35 40 45 Ala Arg Leu Ala Ser Pro Leu Pro Arg Glu Glu Glu IleVal Phe Pro 50 55 60 Glu Lys Leu Asn Gly Ser Val Leu Pro Gly Ser Gly AlaPro Ala Arg 65 70 75 80 Leu Leu Cys Arg Leu Gln Ala Phe Gly Glu Thr LeuLeu Leu Glu Leu 85 90 95 Glu Gln Asp Ser Gly Val Gln Val Glu Gly Leu ThrVal Gln Tyr Leu 100 105 110 Gly Gln Ala Pro Glu Leu Leu Gly Gly Ala GluPro Gly Thr Tyr Leu 115 120 125 Thr Gly Thr Ile Asn Gly Asp Pro Glu SerVal Ala Ser Leu His Trp 130 135 140 Asp Gly Gly Ala Leu Leu Gly Val LeuGln Tyr Arg Gly Ala Glu Leu 145 150 155 160 His Leu Gln Pro Leu Glu GlyGly Thr Pro Asn Ser Ala Gly Gly Pro 165 170 175 Gly Ala His Ile Leu ArgArg Lys Ser Pro Ala Ser Gly Gln Gly Pro 180 185 190 Met Cys Asn Val LysAla Pro Leu Gly Ser Pro Ser Pro Arg Pro Arg 195 200 205 Arg Ala Lys ArgPhe Ala Ser Leu Ser Arg Phe Val Glu Thr Leu Val 210 215 220 Val Ala AspAsp Lys Met Ala Ala Phe His Gly Ala Gly Leu Lys Arg 225 230 235 240 TyrLeu Leu Thr Val Met Ala Ala Ala Ala Lys Ala Phe Lys His Pro 245 250 255Ser Ile Arg Asn Pro Val Ser Leu Val Val Thr Arg Leu Val Ile Leu 260 265270 Gly Ser Gly Glu Glu Gly Pro Gln Val Gly Pro Ser Ala Ala Gln Thr 275280 285 Leu Arg Ser Phe Cys Ala Trp Gln Arg Gly Leu Asn Thr Pro Glu Asp290 295 300 Ser Asp Pro Asp His Phe Asp Thr Ala Ile Leu Phe Thr Arg GlnAsp 305 310 315 320 Leu Cys Gly Val Ser Thr Cys Asp Thr Leu Gly Met AlaAsp Val Gly 325 330 335 Thr Val Cys Asp Pro Ala Arg Ser Cys Ala Ile ValGlu Asp Asp Gly 340 345 350 Leu Gln Ser Ala Phe Thr Ala Ala His Glu LeuGly His Val Phe Asn 355 360 365 Met Leu His Asp Asn Ser Lys Pro Cys IleSer Leu Asn Gly Pro Leu 370 375 380 Ser Thr Ser Arg His Val Met Ala ProVal Met Ala His Val Asp Pro 385 390 395 400 Glu Glu Pro Trp Ser Pro CysSer Ala Arg Phe Ile Thr Asp Phe Leu 405 410 415 Asp Asn Gly Tyr Gly HisCys Leu Leu Asp Lys Pro Glu Ala Pro Leu 420 425 430 His Leu Pro Val ThrPhe Pro Gly Lys Asp Tyr Asp Ala Asp Arg Gln 435 440 445 Cys Gln Leu ThrPhe Gly Pro Asp Ser Arg His Cys Pro Gln Leu Pro 450 455 460 Pro Pro CysAla Ala Leu Trp Cys Ser Gly His Leu Asn Gly His Ala 465 470 475 480 MetCys Gln Thr Lys His Ser Pro Trp Ala Asp Gly Thr Pro Cys Gly 485 490 495Pro Ala Gln Ala Cys Met Gly Gly Arg Cys Leu His Met Asp Gln Leu 500 505510 Gln Asp Phe Asn Ile Pro Gln Ala Gly Gly Trp Gly Pro Trp Gly Pro 515520 525 Trp Gly Asp Cys Ser Arg Thr Cys Gly Gly Gly Val Gln Phe Ser Ser530 535 540 Arg Asp Cys Thr Arg Pro Val Pro Arg Asn Gly Gly Lys Tyr CysGlu 545 550 555 560 Gly Arg Arg Thr Arg Phe Arg Ser Cys Asn Thr Glu AspCys Pro Thr 565 570 575 Gly Ser Ala Leu Thr Phe Arg Glu Glu Gln Cys AlaAla Tyr Asn His 580 585 590 Arg Thr Asp Leu Phe Lys Ser Phe Pro Gly ProMet Asp Trp Val Pro 595 600 605 Arg Tyr Thr Gly Val Ala Pro Gln Asp GlnCys Lys Leu Thr Cys Gln 610 615 620 Ala Arg Ala Leu Gly Tyr Tyr Tyr ValLeu Glu Pro Arg Val Val Asp 625 630 635 640 Gly Thr Pro Cys Ser Pro AspSer Ser Ser Val Cys Val Gln Gly Arg 645 650 655 Cys Ile His Ala Gly CysAsp Arg Ile Ile Gly Ser Lys Lys Lys Phe 660 665 670 Asp Lys Cys Met ValCys Gly Gly Asp Gly Ser Gly Cys Ser Lys Gln 675 680 685 Ser Gly Ser PheArg Lys Phe Arg Tyr Gly Tyr Asn Asn Val Val Thr 690 695 700 Ile Pro AlaGly Ala Thr His Ile Leu Val Arg Gln Gln Gly Asn Pro 705 710 715 720 GlyHis Arg Ser Ile Tyr Leu Ala Leu Lys Leu Pro Asp Gly Ser Tyr 725 730 735Ala Leu Asn Gly Glu Tyr Thr Leu Met Pro Ser Pro Thr Asp Val Val 740 745750 Leu Pro Gly Ala Val Ser Leu Arg Tyr Ser Gly Ala Thr Ala Ala Ser 755760 765 Glu Thr Leu Ser Gly His Gly Pro Leu Ala Gln Pro Leu Thr Leu Gln770 775 780 Val Leu Val Ala Gly Asn Pro Gln Asp Thr Arg Leu Arg Tyr SerPhe 785 790 795 800 Phe Val Pro Arg Pro Thr Pro Ser Thr Pro Arg Pro ThrPro Gln Asp 805 810 815 Trp Leu His Arg Arg Ala Gln Ile Leu Glu Ile LeuArg Arg Arg Pro 820 825 830 Trp Ala Gly Arg Lys 835 3 3 000 4 26 PRT Bostaurus 4 Phe Ala Ser Leu Ser Arg Val Glu Thr Leu Val Val Ala Asp Asp Lys1 5 10 15 Met Ala Ala Phe His Gly Ala Gly Leu Lys 20 25 5 7 PRT Bostaurus 5 Tyr Thr Gly Val Ala Pro Arg 1 5 6 11 PRT Bos taurus 6 Ala LeuGly Tyr Tyr Tyr Val Leu Asp Pro Arg 1 5 10 7 21 DNA Mus musculus 7gggggtggtg tccagttctc c 21 8 23 DNA Mus musculus 8 ggccctggaa agctcttgaagag 23 9 23 DNA Homo sapiens 9 ccccggaatg gtggcaagta ctg 23 10 23 DNAHomo sapiens 10 acccacatct gtctgactcc aaa 23 11 23 DNA Homo sapiens 11ccagttgggc agtcctcagt gtt 23 12 22 DNA Homo sapiens 12 ggtcggtgcggtggttgtag gc 22 13 17 PRT Homo sapiens 13 Cys Ala Ser Leu Ser Arg PheVal Glu Thr Leu Val Val Ala Asp Asp 1 5 10 15 Lys 14 3250 DNA Homosapiens CDS (121)..(2910) 14 tgactcaatc ctgcaagcaa gtgtgtgtgt gtccccatcccccgccccgt taacttcata 60 gcaaataaca aatacccata aagtcccagt cgcgcagcccctccccgcgg gcagcgcact 120 atg ctg ctc ggg tgg gcg tcc ctg ctg ctg tgcgcg ttc cgc ctg ccc 168 Met Leu Leu Gly Trp Ala Ser Leu Leu Leu Cys AlaPhe Arg Leu Pro 1 5 10 15 ctg gcc gcg gtc ggc ccc gcc gcg aca cct gcccag gat aaa gcc ggg 216 Leu Ala Ala Val Gly Pro Ala Ala Thr Pro Ala GlnAsp Lys Ala Gly 20 25 30 cag cct ccg act gct gca gca gcc gcc cag ccc cgccgg cgg cag ggg 264 Gln Pro Pro Thr Ala Ala Ala Ala Ala Gln Pro Arg ArgArg Gln Gly 35 40 45 gag gag gtg cag gag cga gcc gag cct ccc ggc cac ccgcac ccc ctg 312 Glu Glu Val Gln Glu Arg Ala Glu Pro Pro Gly His Pro HisPro Leu 50 55 60 gcg cag cgg cgc agg agc aag ggg ctg gtg cag aac atc gaccaa ctc 360 Ala Gln Arg Arg Arg Ser Lys Gly Leu Val Gln Asn Ile Asp GlnLeu 65 70 75 80 tac tcc ggc ggc ggc aag gtg ggc tac ctc gtc tac gcg ggcggc cgg 408 Tyr Ser Gly Gly Gly Lys Val Gly Tyr Leu Val Tyr Ala Gly GlyArg 85 90 95 agg ttc ctc ttg gac ctg gag cga gat ggt tcg gtg ggc att gctggc 456 Arg Phe Leu Leu Asp Leu Glu Arg Asp Gly Ser Val Gly Ile Ala Gly100 105 110 ttc gtg ccc gca gga ggc ggg acg agt gcg ccc tgg cgc cac cggagc 504 Phe Val Pro Ala Gly Gly Gly Thr Ser Ala Pro Trp Arg His Arg Ser115 120 125 cac tgc ttc tat cgg ggc aca gtg gac gct agt ccc cgc tct ctggct 552 His Cys Phe Tyr Arg Gly Thr Val Asp Ala Ser Pro Arg Ser Leu Ala130 135 140 gtc ttt gac ctc tgt ggg ggt ctc gac ggc ttc ttc gcg gtc aagcac 600 Val Phe Asp Leu Cys Gly Gly Leu Asp Gly Phe Phe Ala Val Lys His145 150 155 160 gcg cgc tac acc cta aag cca ctg ctg cgc gga ccc tgg gcggag gaa 648 Ala Arg Tyr Thr Leu Lys Pro Leu Leu Arg Gly Pro Trp Ala GluGlu 165 170 175 gaa aag ggg cgc gtg tac ggg gat ggg tcc gca cgg atc ctgcac gtc 696 Glu Lys Gly Arg Val Tyr Gly Asp Gly Ser Ala Arg Ile Leu HisVal 180 185 190 tac acc cgc gag ggc ttc agc ttc gag gcc ctg ccg ccg cgcgcc agc 744 Tyr Thr Arg Glu Gly Phe Ser Phe Glu Ala Leu Pro Pro Arg AlaSer 195 200 205 tgc gaa acc ccc gcg tcc aca ccg gag gcc cac gag cat gctccg gcg 792 Cys Glu Thr Pro Ala Ser Thr Pro Glu Ala His Glu His Ala ProAla 210 215 220 cac agc aac ccg agc gga cgc gca gca ctg gcc tcg cag ctcttg gac 840 His Ser Asn Pro Ser Gly Arg Ala Ala Leu Ala Ser Gln Leu LeuAsp 225 230 235 240 cag tcc gct ctc tcg ccc gct ggg ggc tca gga ccg cagacg tgg tgg 888 Gln Ser Ala Leu Ser Pro Ala Gly Gly Ser Gly Pro Gln ThrTrp Trp 245 250 255 cgg cgg cgg cgc cgc tcc atc tcc cgg gcc cgc cag gtggag ctg ctt 936 Arg Arg Arg Arg Arg Ser Ile Ser Arg Ala Arg Gln Val GluLeu Leu 260 265 270 ctg gtg gct gac gcg tcc atg gcg cgg ttg tat ggc cggggc ctg cag 984 Leu Val Ala Asp Ala Ser Met Ala Arg Leu Tyr Gly Arg GlyLeu Gln 275 280 285 cat tac ctg ctg acc ctg gcc tcc atc gcc aat agg ctgtac agc cat 1032 His Tyr Leu Leu Thr Leu Ala Ser Ile Ala Asn Arg Leu TyrSer His 290 295 300 gct agc atc gag aac cac atc cgc ctg gcc gtg gtg aaggtg gtg gtg 1080 Ala Ser Ile Glu Asn His Ile Arg Leu Ala Val Val Lys ValVal Val 305 310 315 320 cta ggc gac aag gac aag agc ctg gaa gtg agc aagaac gct gcc acc 1128 Leu Gly Asp Lys Asp Lys Ser Leu Glu Val Ser Lys AsnAla Ala Thr 325 330 335 aca ctc aag aac ttt tgc aag tgg cag cac caa cacaac cag ctg gga 1176 Thr Leu Lys Asn Phe Cys Lys Trp Gln His Gln His AsnGln Leu Gly 340 345 350 gat gac cat gag gag cac tac gat gca gct atc ctgttt act cgg gag 1224 Asp Asp His Glu Glu His Tyr Asp Ala Ala Ile Leu PheThr Arg Glu 355 360 365 gat tta tgt ggg cat cat tca tgt gac acc ctg ggaatg gca gac gtt 1272 Asp Leu Cys Gly His His Ser Cys Asp Thr Leu Gly MetAla Asp Val 370 375 380 ggg acc ata tgt tct cca gag cgc agc tgt gct gtgatt gaa gac gat 1320 Gly Thr Ile Cys Ser Pro Glu Arg Ser Cys Ala Val IleGlu Asp Asp 385 390 395 400 ggc ctc cac gca gcc ttc act gtg gct cac gaaatc gga cat tta ctt 1368 Gly Leu His Ala Ala Phe Thr Val Ala His Glu IleGly His Leu Leu 405 410 415 ggc ctc tcc cat gac gat tcc aaa ttc tgt gaagag acc ttt ggt tcc 1416 Gly Leu Ser His Asp Asp Ser Lys Phe Cys Glu GluThr Phe Gly Ser 420 425 430 aca gaa gat aag cgc tta atg tct tcc atc cttacc agc att gat gca 1464 Thr Glu Asp Lys Arg Leu Met Ser Ser Ile Leu ThrSer Ile Asp Ala 435 440 445 tct aag ccc tgg tcc aaa tgc act tca gcc accatc aca gaa ttc ctg 1512 Ser Lys Pro Trp Ser Lys Cys Thr Ser Ala Thr IleThr Glu Phe Leu 450 455 460 gat gat ggc cat ggt aac tgt ttg ctg gac ctacca cga aag cag atc 1560 Asp Asp Gly His Gly Asn Cys Leu Leu Asp Leu ProArg Lys Gln Ile 465 470 475 480 ctg ggc ccc gaa gaa ctc cca gga cag acctac gat gcc acc cag cag 1608 Leu Gly Pro Glu Glu Leu Pro Gly Gln Thr TyrAsp Ala Thr Gln Gln 485 490 495 tgc aac ctg aca ttc ggg cct gag tac tccgtg tgt ccc ggc atg gat 1656 Cys Asn Leu Thr Phe Gly Pro Glu Tyr Ser ValCys Pro Gly Met Asp 500 505 510 gtc tgt gct cgc ctg tgg tgt gct gtg gtacgc cag ggc cag atg gtc 1704 Val Cys Ala Arg Leu Trp Cys Ala Val Val ArgGln Gly Gln Met Val 515 520 525 tgt ctg acc aag aag ctg cct gcg gtg gaaggg acg cct tgt gga aag 1752 Cys Leu Thr Lys Lys Leu Pro Ala Val Glu GlyThr Pro Cys Gly Lys 530 535 540 ggg aga atc tgc ctg cag ggc aaa tgt gtggac aaa acc aag aaa aaa 1800 Gly Arg Ile Cys Leu Gln Gly Lys Cys Val AspLys Thr Lys Lys Lys 545 550 555 560 tat tat tca acg tca agc cat ggc aactgg gga tct tgg gga tcc tgg 1848 Tyr Tyr Ser Thr Ser Ser His Gly Asn TrpGly Ser Trp Gly Ser Trp 565 570 575 ggc cag tgt tct cgc tca tgt gga ggagga gtg cag ttt gcc tat cgt 1896 Gly Gln Cys Ser Arg Ser Cys Gly Gly GlyVal Gln Phe Ala Tyr Arg 580 585 590 cac tgt aat aac cct gct ccc aga aacaac gga cgc tac tgc aca ggg 1944 His Cys Asn Asn Pro Ala Pro Arg Asn AsnGly Arg Tyr Cys Thr Gly 595 600 605 aag agg gcc atc tac cgc tcc tgc agtctc atg ccc tgc cca ccc aat 1992 Lys Arg Ala Ile Tyr Arg Ser Cys Ser LeuMet Pro Cys Pro Pro Asn 610 615 620 ggt aaa tca ttt cgt cat gaa cag tgtgag gcc aaa aat ggc tat cag 2040 Gly Lys Ser Phe Arg His Glu Gln Cys GluAla Lys Asn Gly Tyr Gln 625 630 635 640 tct gat gca aaa gga gtc aaa actttt gtg gaa tgg gtt ccc aaa tat 2088 Ser Asp Ala Lys Gly Val Lys Thr PheVal Glu Trp Val Pro Lys Tyr 645 650 655 gca ggt gtc ctg cca gcg gat gtgtgc aag ctg acc tgc aga gcc aag 2136 Ala Gly Val Leu Pro Ala Asp Val CysLys Leu Thr Cys Arg Ala Lys 660 665 670 ggc act ggc tac tat gtg gta ttttct cca aag gtg acc gat ggc act 2184 Gly Thr Gly Tyr Tyr Val Val Phe SerPro Lys Val Thr Asp Gly Thr 675 680 685 gaa tgt agg ccg tac agt aat tccgtc tgc gtc cgg ggg aag tgt gtg 2232 Glu Cys Arg Pro Tyr Ser Asn Ser ValCys Val Arg Gly Lys Cys Val 690 695 700 aga act ggc tgt gac ggc atc attggc tca aag ctg cag tat gac aag 2280 Arg Thr Gly Cys Asp Gly Ile Ile GlySer Lys Leu Gln Tyr Asp Lys 705 710 715 720 tgc gga gta tgt gga gga gacaac tcc agc tgt aca aag att gtt gga 2328 Cys Gly Val Cys Gly Gly Asp AsnSer Ser Cys Thr Lys Ile Val Gly 725 730 735 acc ttt aat aag aaa agt aagggt tac act gac gtg gtg agg att cct 2376 Thr Phe Asn Lys Lys Ser Lys GlyTyr Thr Asp Val Val Arg Ile Pro 740 745 750 gaa ggg gca acc cac ata aaagtt cga cag ttc aaa gcc aaa gac cag 2424 Glu Gly Ala Thr His Ile Lys ValArg Gln Phe Lys Ala Lys Asp Gln 755 760 765 act aga ttc act gcc tat ttagcc ctg aaa aag aaa aac ggt gag tac 2472 Thr Arg Phe Thr Ala Tyr Leu AlaLeu Lys Lys Lys Asn Gly Glu Tyr 770 775 780 ctt atc aat gga aag tac atgatc tcc act tca gag act atc att gac 2520 Leu Ile Asn Gly Lys Tyr Met IleSer Thr Ser Glu Thr Ile Ile Asp 785 790 795 800 atc aat gga aca gtc atgaac tat agc ggt tgg agc cac agg gat gac 2568 Ile Asn Gly Thr Val Met AsnTyr Ser Gly Trp Ser His Arg Asp Asp 805 810 815 ttc ctg cat ggc atg ggctac tct gcc acg aag gaa att cta ata gtg 2616 Phe Leu His Gly Met Gly TyrSer Ala Thr Lys Glu Ile Leu Ile Val 820 825 830 cag att ctt gca aca gacccc act aaa cca tta gat gtc cgt tat agc 2664 Gln Ile Leu Ala Thr Asp ProThr Lys Pro Leu Asp Val Arg Tyr Ser 835 840 845 ttt ttt gtt ccc aag aagtcc act cca aaa gta aac tct gtc act agt 2712 Phe Phe Val Pro Lys Lys SerThr Pro Lys Val Asn Ser Val Thr Ser 850 855 860 cat ggc agc aat aaa gtggga tca cac act tcg cag ccg cag tgg gtc 2760 His Gly Ser Asn Lys Val GlySer His Thr Ser Gln Pro Gln Trp Val 865 870 875 880 acg ggc cca tgg ctcgcc tgc tct agg acc tgt gac aca ggt tgg cac 2808 Thr Gly Pro Trp Leu AlaCys Ser Arg Thr Cys Asp Thr Gly Trp His 885 890 895 acc aga acg gtg cagtgc cag gat gga aac cgg aag tta gca aaa gga 2856 Thr Arg Thr Val Gln CysGln Asp Gly Asn Arg Lys Leu Ala Lys Gly 900 905 910 tgt cct ctc tcc caaagg cct tct gcg ttt aag caa tgc ttg ttg aag 2904 Cys Pro Leu Ser Gln ArgPro Ser Ala Phe Lys Gln Cys Leu Leu Lys 915 920 925 aaa tgt tagcctgtggttatgatctt atgcacaaag ataactggag gattcagcac 2960 Lys Cys 930 cgatgcagtcgtggtgaaca ggaggtctac ctaacgcaca gaaagtcatg cttcagtgac 3020 attgtcaacaggagtccaat tatgggcaga atctgctctc tgtgaccaaa agaggatgtg 3080 cactgcttcacgtgacagtg gtgaccttgc aatatagaaa aacttgggag ttattgaaca 3140 tcccctgggattacaagaaa cactgatgaa tgttaaatca ggggacattt gaagatggca 3200 gaactgtctcccccttgtca cctacctctg aatagaatgt ctttaatggt 3250 15 930 PRT Homo sapiens15 Met Leu Leu Gly Trp Ala Ser Leu Leu Leu Cys Ala Phe Arg Leu Pro 1 510 15 Leu Ala Ala Val Gly Pro Ala Ala Thr Pro Ala Gln Asp Lys Ala Gly 2025 30 Gln Pro Pro Thr Ala Ala Ala Ala Ala Gln Pro Arg Arg Arg Gln Gly 3540 45 Glu Glu Val Gln Glu Arg Ala Glu Pro Pro Gly His Pro His Pro Leu 5055 60 Ala Gln Arg Arg Arg Ser Lys Gly Leu Val Gln Asn Ile Asp Gln Leu 6570 75 80 Tyr Ser Gly Gly Gly Lys Val Gly Tyr Leu Val Tyr Ala Gly Gly Arg85 90 95 Arg Phe Leu Leu Asp Leu Glu Arg Asp Gly Ser Val Gly Ile Ala Gly100 105 110 Phe Val Pro Ala Gly Gly Gly Thr Ser Ala Pro Trp Arg His ArgSer 115 120 125 His Cys Phe Tyr Arg Gly Thr Val Asp Ala Ser Pro Arg SerLeu Ala 130 135 140 Val Phe Asp Leu Cys Gly Gly Leu Asp Gly Phe Phe AlaVal Lys His 145 150 155 160 Ala Arg Tyr Thr Leu Lys Pro Leu Leu Arg GlyPro Trp Ala Glu Glu 165 170 175 Glu Lys Gly Arg Val Tyr Gly Asp Gly SerAla Arg Ile Leu His Val 180 185 190 Tyr Thr Arg Glu Gly Phe Ser Phe GluAla Leu Pro Pro Arg Ala Ser 195 200 205 Cys Glu Thr Pro Ala Ser Thr ProGlu Ala His Glu His Ala Pro Ala 210 215 220 His Ser Asn Pro Ser Gly ArgAla Ala Leu Ala Ser Gln Leu Leu Asp 225 230 235 240 Gln Ser Ala Leu SerPro Ala Gly Gly Ser Gly Pro Gln Thr Trp Trp 245 250 255 Arg Arg Arg ArgArg Ser Ile Ser Arg Ala Arg Gln Val Glu Leu Leu 260 265 270 Leu Val AlaAsp Ala Ser Met Ala Arg Leu Tyr Gly Arg Gly Leu Gln 275 280 285 His TyrLeu Leu Thr Leu Ala Ser Ile Ala Asn Arg Leu Tyr Ser His 290 295 300 AlaSer Ile Glu Asn His Ile Arg Leu Ala Val Val Lys Val Val Val 305 310 315320 Leu Gly Asp Lys Asp Lys Ser Leu Glu Val Ser Lys Asn Ala Ala Thr 325330 335 Thr Leu Lys Asn Phe Cys Lys Trp Gln His Gln His Asn Gln Leu Gly340 345 350 Asp Asp His Glu Glu His Tyr Asp Ala Ala Ile Leu Phe Thr ArgGlu 355 360 365 Asp Leu Cys Gly His His Ser Cys Asp Thr Leu Gly Met AlaAsp Val 370 375 380 Gly Thr Ile Cys Ser Pro Glu Arg Ser Cys Ala Val IleGlu Asp Asp 385 390 395 400 Gly Leu His Ala Ala Phe Thr Val Ala His GluIle Gly His Leu Leu 405 410 415 Gly Leu Ser His Asp Asp Ser Lys Phe CysGlu Glu Thr Phe Gly Ser 420 425 430 Thr Glu Asp Lys Arg Leu Met Ser SerIle Leu Thr Ser Ile Asp Ala 435 440 445 Ser Lys Pro Trp Ser Lys Cys ThrSer Ala Thr Ile Thr Glu Phe Leu 450 455 460 Asp Asp Gly His Gly Asn CysLeu Leu Asp Leu Pro Arg Lys Gln Ile 465 470 475 480 Leu Gly Pro Glu GluLeu Pro Gly Gln Thr Tyr Asp Ala Thr Gln Gln 485 490 495 Cys Asn Leu ThrPhe Gly Pro Glu Tyr Ser Val Cys Pro Gly Met Asp 500 505 510 Val Cys AlaArg Leu Trp Cys Ala Val Val Arg Gln Gly Gln Met Val 515 520 525 Cys LeuThr Lys Lys Leu Pro Ala Val Glu Gly Thr Pro Cys Gly Lys 530 535 540 GlyArg Ile Cys Leu Gln Gly Lys Cys Val Asp Lys Thr Lys Lys Lys 545 550 555560 Tyr Tyr Ser Thr Ser Ser His Gly Asn Trp Gly Ser Trp Gly Ser Trp 565570 575 Gly Gln Cys Ser Arg Ser Cys Gly Gly Gly Val Gln Phe Ala Tyr Arg580 585 590 His Cys Asn Asn Pro Ala Pro Arg Asn Asn Gly Arg Tyr Cys ThrGly 595 600 605 Lys Arg Ala Ile Tyr Arg Ser Cys Ser Leu Met Pro Cys ProPro Asn 610 615 620 Gly Lys Ser Phe Arg His Glu Gln Cys Glu Ala Lys AsnGly Tyr Gln 625 630 635 640 Ser Asp Ala Lys Gly Val Lys Thr Phe Val GluTrp Val Pro Lys Tyr 645 650 655 Ala Gly Val Leu Pro Ala Asp Val Cys LysLeu Thr Cys Arg Ala Lys 660 665 670 Gly Thr Gly Tyr Tyr Val Val Phe SerPro Lys Val Thr Asp Gly Thr 675 680 685 Glu Cys Arg Pro Tyr Ser Asn SerVal Cys Val Arg Gly Lys Cys Val 690 695 700 Arg Thr Gly Cys Asp Gly IleIle Gly Ser Lys Leu Gln Tyr Asp Lys 705 710 715 720 Cys Gly Val Cys GlyGly Asp Asn Ser Ser Cys Thr Lys Ile Val Gly 725 730 735 Thr Phe Asn LysLys Ser Lys Gly Tyr Thr Asp Val Val Arg Ile Pro 740 745 750 Glu Gly AlaThr His Ile Lys Val Arg Gln Phe Lys Ala Lys Asp Gln 755 760 765 Thr ArgPhe Thr Ala Tyr Leu Ala Leu Lys Lys Lys Asn Gly Glu Tyr 770 775 780 LeuIle Asn Gly Lys Tyr Met Ile Ser Thr Ser Glu Thr Ile Ile Asp 785 790 795800 Ile Asn Gly Thr Val Met Asn Tyr Ser Gly Trp Ser His Arg Asp Asp 805810 815 Phe Leu His Gly Met Gly Tyr Ser Ala Thr Lys Glu Ile Leu Ile Val820 825 830 Gln Ile Leu Ala Thr Asp Pro Thr Lys Pro Leu Asp Val Arg TyrSer 835 840 845 Phe Phe Val Pro Lys Lys Ser Thr Pro Lys Val Asn Ser ValThr Ser 850 855 860 His Gly Ser Asn Lys Val Gly Ser His Thr Ser Gln ProGln Trp Val 865 870 875 880 Thr Gly Pro Trp Leu Ala Cys Ser Arg Thr CysAsp Thr Gly Trp His 885 890 895 Thr Arg Thr Val Gln Cys Gln Asp Gly AsnArg Lys Leu Ala Lys Gly 900 905 910 Cys Pro Leu Ser Gln Arg Pro Ser AlaPhe Lys Gln Cys Leu Leu Lys 915 920 925 Lys Cys 930 16 42 PRT Homosapiens 16 Ser Ile Ser Arg Ala Arg Gln Val Glu Leu Leu Leu Val Ala AspAla 1 5 10 15 Ser Met Ala Arg Met Tyr Gly Arg Gly Leu Gln His Tyr LeuLeu Thr 20 25 30 Leu Ala Ser Ile Ala Asn Lys Leu Tyr Phe 35 40 17 23 DNAMus musculus 17 cggccacgac cctcaagaac ttt 23 18 25 DNA Mus musculus 18gcatggaggc catcatcttc aatca 25 19 22 DNA Homo sapiens 19 gggaggatttatgtgggcat ca 22 20 23 DNA Homo sapiens 20 gtgcatttgg accagggctt aga 2321 13 PRT Artificial Sequence Synthesized peptide. 21 Ser Ile Ser ArgAla Arg Gln Val Glu Leu Leu Xaa Cys 1 5 10

What is claimed:
 1. An isolated nucleic acid molecule comprising thenucleic acid sequence of SEQ ID NO:1.
 2. An expression vector comprisingthe nucleic acid molecule of claim
 1. 3. A host cell transfected ortransformed with the expression vector of claim
 2. 4. A method ofproducing an ADMP which comprises culturing the host cell of claim 3under conditions suitable for expressing the nucleic acid molecule andfor translation of the resulting mRNA and